Three-step pretargeting methods using improved biotin-active agent

ABSTRACT

Methods, compounds, compositions and kits that relate to pretargeted delivery of diagnostic and therapeutic agents are disclosed. In particular, three-step pretargeting methods are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of pending PCT PatentApplication No. PCT/US93/05406, filed Jun. 7, 1993 and designating theUnited States, which, in turn, is a continuation-in-part of pending U.S.patent application Ser. No. 07/995,383, filed Dec. 23, 1992, nowabandoned, which is, in turn, a continuation-in-part of pending U.S.patent application Ser. No. 07/895,588, filed Jun. 9, 1992, now U.S.Pat. No. 5,283,342.

TECHNICAL FIELD

The present invention relates to methods, compounds, compositions andkits useful for delivering to a target site a targeting moiety that isconjugated to one member of a ligand/anti-ligand pair. Afterlocalization and clearance of the targeting moiety conjugate, direct orindirect binding of a diagnostic or therapeutic agent conjugate at thetarget site occurs. Methods for radiometal labeling of biotin and forimproved radiohalogenation of biotin, as well as the related compounds,are also disclosed.

SUMMARY OF THE INVENTION

The present invention describes three-step pretargeting diagnostic andtherapeutic methods. Three-step pretargeting protocols featureadministration of a targeting moiety-ligand conjugate, which is allowedto localize at a target site and to dilute in the circulation.Subsequently administered anti-ligand binds to the targetingmoiety-ligand conjugate in both blood and at a target site and clearsunbound antibody-ligand conjugate from the blood. A diagnostic ortherapeutic agent-ligand conjugate that exhibits rapid whole bodyclearance is then administered and binds to the targetingmoiety-ligand-anti-ligand localized at a target site, therebyconstituting the third target site-localized component in the protocol.

Preferred three-step pretargeting methods of the present inventionemploy biotin/avidin as the ligand/anti-ligand binding pair. Thesepreferred three-step pretargeting methods involve the administration ofbiotin conjugated to therapeutic or diagnostic radionuclides or otheractive agents such as chemotherapeutic drugs, anti-tumor agents such ascytokines and the like. Y-90-DOTA-biotin conjugates are particularlypreferred in the practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates blood clearance of biotinylated antibody followingintravenous administration of avidin.

FIG. 2 depicts radiorhenium tumor uptake in a three-step pretargetingprotocol, as compared to administration of radiolabeled antibody(conventional means involving antibody that is covalently linked tochelated radiorhenium).

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to set forthdefinitions of certain terms to be used within the disclosure.

Targeting Moiety

A molecule that binds to a defined population of cells. The targetingmoiety may bind a receptor, an enzymatic substrate, an antigenicdeterminant, or other binding site present on the target cellpopulation. Antibody is used throughout the specification as aprototypical example of .a targeting moiety.

Ligand/Anti-Ligand Pair

A complementary/anti-complementary set of molecules that demonstratespecific binding, generally of relatively high affinity. Exemplaryligand/anti-ligand pairs include zinc finger protein/dsDNA fragment,enzyme/inhibitor, hapten/antibody, ligand/receptor, and biotin/avidin.Biotin/avidin is used throughout the specification as a prototypicalexample of a ligand/anti-ligand pair.

Anti-Ligand

As defined herein, an "anti-ligand" demonstrates high affinity, andpreferably, multivalent binding of the complementary ligand. Preferably,the anti-ligand is large enough to avoid rapid renal clearance, andcontains sufficient multivalency to accomplish crosslinking andaggregation of targeting moiety-ligand conjugates. Univalentanti-ligands also find utility in the practice of the present invention.

Avidin

As defined herein, "avidin" includes avidin, streptavidin andderivatives and analogs thereof that are capable of high affinity,multivalent or univalent binding of biotin.

Ligand

As defined herein, a "ligand" is a relatively small, soluble moleculethat exhibits rapid serum, blood and/or whole body clearance whenadministered intravenously in an animal or human.

Active Agent

A diagnostic or therapeutic agent ("the payload"), includingradionuclides, drugs, anti-tumor agents, toxins and the like.

N_(x) S_(y) Chelates

As defined herein, the term "N_(x) S_(y) chelates" includes bifunctionalchelators that are capable of (i) coordinately binding a metal orradiometal and (ii) covalently attaching to a targeting moiety.Particularly preferred N_(x) S_(y) chelates have N₂ S₂ and N₃ S cores.Exemplary N_(x) S_(y) chelates are described in Fritzberg et al., Proc.Natl. Acad. Sci. U.S.A. 85:4024-29, 1988; in Weber et al., Bioconj.Chem. 1:431-37, 1990; and in the references cited therein, for instance.

Pretargeting

As defined herein, pretargeting involves target site localization of atargeting moiety that is conjugated with one member of aligand/anti-ligand pair; after a time period sufficient for optimaltarget-to-non-target accumulation of this targeting moiety conjugate,active agent conjugated to the opposite member of the ligand/anti-ligandpair is administered and is bound (directly or indirectly) to thetargeting moiety conjugate at the target site (two-step pretargeting).Three-step pretargeting protocols are also provided by the presentinvention, involving, for example, administration of targetingmoiety-ligand, administration of anti-ligand to clear circulatingtargeting moiety-ligand and to localize to previously target-localizedtargeting moiety ligand, and administration of active agent-ligand.

Conjugate

A conjugate encompasses chemical conjugates (covalently ornon-covalently bound), fusion proteins and the like.

A recognized disadvantage associated with in vivo administration oftargeting moiety-radioisotopic conjugates for imaging or therapy islocalization of the attached radioactive agent at both non-target andtarget sites. Until the administered radiolabeled conjugate clears fromthe circulation, normal organs and tissues are transitorily exposed tothe attached radioactive agent. For instance, radiolabeled wholeantibodies that are administered in vivo exhibit relatively slow bloodclearance; maximum target site localization generally occurs 1-3 dayspost-administration. Generally, the longer the clearance time of theconjugate from the circulation, the greater the radioexposure ofnon-target organs.

These characteristics are particularly problematic with humanradioimmunotherapy. In human clinical trials, the long circulatinghalf-life of radioisotope bound to whole antibody causes relativelylarge doses of radiation to be delivered to the whole body. Inparticular, the bone marrow, which is very radiosensitive, is thedose-limiting organ of non-specific toxicity.

In order to decrease radioisotope exposure of non-target tissue,potential targeting moieties generally have been screened to identifythose that display minimal non-target reactivity, while retaining targetspecificity and reactivity. By reducing non-target exposure (and adversenon-target localization and/or toxicity), increased doses of aradiotherapeutic conjugate may be administered; moreover, decreasednon-target accumulation of a radiodiagnostic conjugate leads to improvedcontrast between background and target.

Therapeutic drugs, administered alone or as targeted conjugates, areaccompanied by similar disadvantages. Again, the goal is administrationof the highest possible concentration of drug (to maximize exposure oftarget tissue), while remaining below the threshold of unacceptablenormal organ toxicity (due to non-target tissue exposure). Unlikeradioisotopes, however, therapeutic drugs need to be taken into a targetcell to exert a cytotoxic effect. In the case of targetingmoiety-therapeutic drug conjugates, it would be advantageous to combinethe relative target specificity of a targeting moiety with a means forenhanced target cell internalization of the targeting moiety-drugconjugate.

In contrast, enhanced target cell internalization is disadvantageous ifone administers diagnostic agent-targeting moiety conjugates.Internalization of diagnostic conjugates results in cellular catabolismand degradation of the conjugate. Upon degradation, small adducts of thediagnostic agent or the diagnostic agent per se may be released from thecell, thus eliminating the ability to detect the conjugate in atarget-specific manner.

One method for reducing non-target tissue exposure to a diagnostic ortherapeutic agent involves "pretargeting" the targeting moiety at atarget site, and then subsequently administering a rapidly clearingdiagnostic or therapeutic agent conjugate that is capable of binding tothe "pretargeted" targeting moiety at the target site. A description ofsome embodiments of the pretargeting technique may be found in U.S. Pat.No. 4,863,713 (Goodwin et al.).

A typical pretargeting approach ("three-step") is schematically depictedbelow. ##STR1## Briefly, this three-step pretargeting protocol featuresadministration of an antibody-ligand conjugate, which is allowed tolocalize at a target site and to dilute in the circulation. Subsequentlyadministered anti-ligand binds to the antibody-ligand conjugate andclears unbound antibody-ligand conjugate from the blood. Preferredanti-ligands are large and contain sufficient multivalency to accomplishcrosslinking and aggregation of circulating antibody-ligand conjugates.The clearing by anti-ligand is probably attributable to anti-ligandcrosslinking and/or aggregation of antibody-ligand conjugates that arecirculating in the blood, which leads to complex/aggregate clearance bythe recipient's RES. It is preferred that the ligand-anti-ligand pairdisplays relatively high affinity binding.

A diagnostic or therapeutic agent-ligand conjugate that exhibits rapidwhole body clearance is then administered. When the circulation bringsthe active agent-ligand conjugate in proximity to the target cell-boundantibody-ligand-anti-ligand complex, anti-ligand binds the circulatingactive agent-ligand conjugate and produces anantibody-ligand:anti-ligand:ligand-active agent "sandwich" at the targetsite. Because the diagnostic or therapeutic agent is attached to arapidly clearing ligand (rather than antibody, antibody fragment orother slowly clearing targeting moiety), this technique promisesdecreased non-target exposure to the active agent.

Alternate pretargeting methods eliminate the step of parenterallyadministering an anti-ligand clearing agent. These "two-step" proceduresfeature targeting moiety-ligand or targeting moiety-anti-ligandadministration, followed by administration of active agent conjugated tothe opposite member of the ligand-anti-ligand pair.

The present invention provides methods for radiolabeling biotin withtechnetium-99m, rhenium-186 and rhenium-188 are disclosed. Previously,biotin derivatives were radiolabeled with indium-111 for use inpretargeted immunoscintigraphy (for instance, Virzi et al., Nucl. Med.Biol. 18:719-26, 1991; Kalofonos et al., J. Nucl. Med. 31: 1791-96,1990; Paganelli et al., Canc. Res., 51:5960-66, 1991). However, ^(99m)Tc is a particularly preferred radionuclide for immunoscintigraphy dueto (i) low cost, (ii) convenient supply and (iii) favorable nuclearproperties. Rhenium-186 displays chelating chemistry very similar to^(99m) Tc, and is considered to be an excellent therapeutic radionuclide(i.e., a 3.7 day half-life and 1.07 MeV maximum particle that is similarto ¹³¹ I). Therefore, the claimed methods for technetium and rheniumradiolabeling of biotin numerous advantages.

The present invention is also directed to radiolabeling with yttrium-90,lutetium-177, sumarium-153, and other appropriate +3 metals. Y-90 is aparticularly preferred beta particle emitting radionuclide for therapy,because it exhibits favorable nuclear properties including high specificactivity, long path length with respect to deposition of radiation intissue, high equilibrium dose constant and favorable half-lifeproperties. More specifically, the beta emission of Y-90 (Beta_(av)=0.937 MeV) is one of the most energetic of all beta emitters. The X₉₀value of Y-90 is 5.34 mm (i.e., 90% of the energy emitted from a pointsource is absorbed in a sphere of 5.34 mm radius). Y-90 has a highequilibrium dose constant or mean energy/nuclear transition, Delta=1.99Rad-gram/microcurie-hour, and a 64 hour half-life suitable for targetedtherapy. Y-90 can be manufactured at high specific activity and isavailable as a generator product. Specific advantages of Y-90 are (1)that it has the capability to kill neighboring target Cells not directlytargeted by the pretargeted targeting moiety-ligand or targetingmoiety-anti-ligand conjugate and (2) that more radiation is depositedper microcurie localized than for other beta emitters of lower meanparticle energy (provided that a sufficiently large target volume isavailable).

Lu-177 is a particularly preferred radionuclide for targeted nuclidetherapy, since it has a moderately energetic beta emission (Beta_(av)=0.140 MeV); it is available in high specific activity; itsradiochemical production is efficient; it emits two gammas of idealenergy and abundance for imaging (208 keV, 11% and 113 keV, 7%); and ithas a relatively long half-life (161 hours). The X₉₀ for Lu-177 is 0.31mm, i.e., 90% of the energy emitted form a point source is absorbed in asphere of radius 0.31 mm. Lu-177 has an equilibrium dose constant ormean energy/nuclear transition of 0.31 Rad-gram/microcuries-hour and anadequate half-life to serve as a targeted therapeutic radionuclide.Specific advantages of Lu-177 are (1) that its emitted energy isefficiently absorbed in smaller targeted tumor volumes such asmetastatic tumor foci or involved lymph nodes and (2) that its longphysical half-life makes optimal use of the tumor retention property ofthe pretargeting delivery method. Lu-177 has the additional advantage ofbeing imagable by commonly available nuclear medicine cameras.

The "targeting moiety" of the present invention binds to a definedtarget cell population, such as tumor cells. Preferred targetingmoieties useful in this regard include antibody and antibody fragments,peptides, and hormones. Proteins corresponding to known cell surfacereceptors (including low density lipoproteins, transferrin and insulin),fibrinolytic enzymes, anti-HER2, platelet binding proteins such asannexins, and biological response modifiers (including interleukin,interferon, erythropoietin and colony-stimulating factor) are alsopreferred targeting moieties. Also, anti-EGF receptor antibodies, whichinternalize following binding to the EGF receptor and which traffic tothe nucleus, are preferred targeting moieties for use in the presentinvention to facilitate delivery of Auger emitters and nucleus bindingdrugs to target cell nuclei. Oligonucleotides, e.g., antisenseoligonucleotides that are complementary to portions of target cellnucleic acids (DNA or RNA), are also useful as targeting moieties in thepractice of the present invention. Oligonucleotides binding to cellsurfaces are also useful. Analogs of the above-listed targeting moietiesthat retain the capacity to bind to a defined target cell population mayalso be used within the claimed invention. In addition, synthetictargeting moieties may be designed.

Functional equivalents of the aforementioned molecules are also usefulas targeting moieties of the present invention. One targeting moietyfunctional equivalent is a "mimetic" compound, an organic chemicalconstruct designed to mimic the proper configuration and/or orientationfor targeting moiety-target cell binding. Another targeting moietyfunctional equivalent is a short polypeptide designated as a "minimal"polypeptide, constructed using computer-assisted molecular modeling andmutants having altered binding affinity, which minimal polypeptidesexhibit the binding affinity of the targeting moiety.

Preferred targeting moieties of the present invention are antibodies(polyclonal or monoclonal), peptides, oligonucleotides or the like.Polyclonal antibodies useful in the practice of the present inventionare polyclonal (Vial and Callahan, Univ. Mich. Med. Bull., 20: 284-6,1956), affinity-purified polyclonal or fragments thereof (Chao et al.,Res. Comm. in Chem. Path. & Pharm., 9: 749-61, 1974).

Monoclonal antibodies useful in the practice of the present inventioninclude whole antibody and fragments thereof. Such monoclonal antibodiesand fragments are producible in accordance with conventional techniques,such as hybridoma synthesis, recombinant DNA techniques and proteinsynthesis. Useful monoclonal antibodies and fragments may be derivedfrom any species (including humans) or may be formed as chimericproteins which employ sequences from more than one species. See,generally, Kohler and Milstein, Nature, 256: 495-97, 1975; Eur. J.Immunol., 6: 511-19, 1976.

Human monoclonal antibodies and "humanized" murine antibodies are alsouseful as targeting moieties in accordance with the present invention.Human monoclonal antibodies may be obtained from human serum, fromhybrid mice or other mammals having a functional human immune system,using hybridoma technology, or the like. Also, a murine monoclonalantibody, for example, may be "humanized" by genetically recombining thenucleotide sequence encoding the murine Fv region (i.e., containing theantigen binding site which antibodies are also known as chimericantibodies) or the complementarity determining regions thereof with thenucleotide sequence encoding a human constant domain region and an Fcregion, e.g., in a manner similar to that disclosed in European PatentApplication No. 0,411,893 A2. Some additional murine residues may alsobe retained within the human variable region framework domains to ensureproper target site binding characteristics. Humanized targeting moietiesare recognized to decrease the immunoreactivity of the antibody orpolypeptide in the host recipient, permitting an increase in thehalf-life and a reduction in the possibility of adverse immunereactions.

Types of active agents (diagnostic or therapeutic) useful herein includetoxins, drugs, anti-tumor agents, and radionuclides. Several of thepotent toxins useful within the present invention consist of an A and aB chain. The A chain is the cytotoxic portion and the B chain is thereceptor-binding portion of the intact toxin molecule (holotoxin).Because toxin B chain may mediate non-target cell binding, it is oftenadvantageous to conjugate only the toxin A chain to a targeting protein.However, while elimination of the toxin B chain decreases non-specificcytotoxicity, it also generally leads to decreased potency of the toxinA chain-targeting protein conjugate, as compared to the correspondingholotoxin-targeting protein conjugate.

Preferred toxins in this regard include holotoxins, such as abrin,ricin, modeccin, Pseudomonas exotoxin A, Diphtheria toxin, pertussistoxin and Shiga toxin; and A chain or "A chain-like" molecules, such asricin A chain, abrin A chain, modeccin A chain, the enzymatic portion ofPseudomonas exotoxin A, Diphtheria toxin A chain, the enzymatic portionof pertussis toxin, the enzymatic portion of Shiga toxin, gelonin,pokeweed antiviral protein, saporin, tritin, barley toxin and snakevenom peptides. Ribosomal inactivating proteins (RIPs), naturallyoccurring protein synthesis inhibitors that lack translocating andcell-binding ability, are also suitable for use herein. Extremely highlytoxic toxins, such as palytoxin and the like, are also contemplated foruse in the practice of the present invention.

Charge modification of proteinaceous targeting moieties and conjugatescontaining such targeting moieties and diagnostically or therapeuticallyactive agents is discussed in published European Patent Application No.EP 329,184. Preferred charge modification in accordance with the presentinvention involves treatment of a proteinaceous active agent with aanion-forming reagent to provide a charge-modified moiety or conjugateexhibiting an acidic shift in isoelectric point. Preferably, the shiftin isoelectric point is one-tenth of a pH unit or greater. Generally,charge-modified proteins exhibit a serum half-life that is at least 10%greater than the half-life of native proteins. A 50% or greater increasein half-life is not uncommon following charge modification to a protein.

Anion-forming agents useful in the practice of the present invention arestructured to react with functional groups of the protein to becharge-modified and incorporate a negatively charged group to impart anacidic shift in the pI of the protein to be charge-modified. Preferredanion-forming agents useful in the practice of the present invention arestructured to react with primary amines on lysine residues of theprotein to be charge modified. Such anion-forming agents include activeesters (carboxylic and imide), maleimides and anhydrides. Preferredactive esters include N-hydroxysuccinimidyl, thiophenyl,2,3,5,6-tetrafluorophenyl, and 2,3,5,6,-tetrafluorothiophenyl esters.Derivatization of other protein residues may also be employed in thepractice of the present invention (e.g., derivatization of arginineresidues with glyoxal, phenyl glyoxal or cyclohexanedione). Negativelycharged groups which may be used to impart an acidic shift toproteinaceous active agents include phosphates, phosphonates, sulfates,nitrates, borates, silicates, carbonates, and carboxyl groups such asnative carboxyl groups or carboxyl groups generated from an anhydrideduring the reaction of the anion-forming agent with the protein.

Useful anion-forming agents include compounds incorporating an anhydrideand/or at least one COOH group, such as succinic anhydride, other cyclicacid anhydrides, phthalic anhydride, maleic anhydride, N-ethyl maleimidesubstituted with carboxyl groups, aliphatic anhydrides (e.g., aceticanhydride), aromatic anhydrides, pH-reversible anhydrides (e.g.,citraconic anhydride and dimethyl maleic anhydride), alpha halo acidssuch as bromoacetate and iodoacetate, and diacids or triacidssubstituted with a functional group that reacts with an amino acid on aprotein to be charge-modified.

For example, succinic anhydride is dissolved in DMSO or another dryorganic solvent at a concentration of 40 mg per 200 microliters. Thissuccinic anhydride solution (or a dilution thereof up to 2.5 ml inanhydrous DMSO, 1.73×10⁻² M) is added to a protein (e.g., holotoxin ortoxin domain or conjugate containing one or more of these components)solution (e.g., 3-5 mg/ml in carbonate/bicarbonate buffer, pH 8.5-9.0)at molar ratios of succinic anhydride to protein of 1:5, 1:10 and 1:25(with higher molar ratios preferred). The reaction is carried out atroom temperature for 15-30 minutes. After reaction completion, succinicacid is removed by ultrafiltration or by gel filtration. The degree ofisoelectric shift is determined by isoelectric focusing. The toxicity ofcharge-modified active agents is tested in accordance with knownprocedures for toxicity testing.

Preferred drugs suitable for use herein include conventionalchemotherapeutics, such as vinblastine, doxorubicin, bleomycin,methotrexate, 5-fluorouracil, 6-thioguanine, cytarabine,cyclophosphamide and cis-platinum, as well as other conventionalchemotherapeutics as described in Cancer: Principles and Practice ofOncology, 2d ed., V. T. DeVita, Jr., S. Hellman, S. A. Rosenberg, J.B.Lippincott Co., Philadelphia, Pa., 1985, Chapter 14. A particularlypreferred drug within the present invention is a trichothecene.

Trichothecenes are drugs produced by soil fungi of the class Fungiimperfecti or isolated from Baccharus megapotamica (Bamburg, J. R. Proc.Molec. Subcell. Biol. 8:41-110, 1983; Jarvis & Mazzola, Acc. Chem. Res.15:338-395, 1982). They appear to be the most toxic molecules thatcontain only carbon, hydrogen and oxygen (Tamm, C. Fortschr. Chem. Org.Naturst. 31:61-117, 1974). They are all reported to act at the level ofthe ribosome as inhibitors of protein synthesis at the initiation,elongation, or termination phases.

There are two broad classes of trichothecenes: those that have only acentral sesquiterpenoid structure and those that have an additionalmacrocyclic ring (simple and macrocyclic trichothecenes, respectively).The simple trichothecenes may be subdivided into three groups (i.e.,Group A, B, and C) as described in U.S. Pat. Nos. 4,744,981 and4,906,452 (incorporated herein by reference). Representative examples ofGroup A simple trichothecenes include: Scirpene, Roridin C,dihydrotrichothecene, Scirpen-4, 8-diol, Verrucarol, Scirpentriol, T-2tetraol, pentahydroxyscirpene, 4-deacetylneosolaniol, trichodermin,deacetylcalonectrin, calonectrin, diacetylverrucarol,4-monoacetoxyscirpenol, 4,15-diacetoxyscirpenol,7-hydroxydiacetoxyscirpenol, 8-hydroxydiacetoxy-scirpenol (Neosolaniol),7,8-dihydroxydiacetoxyscirpenol, 7-hydroxy-8-acetyldiacetoxyscirpenol,8-acetylneosolaniol, NT-1, NT-2, HT-2, T-2, and acetyl T-2 toxin.Representative examples of Group B simple trichothecenes include:Trichothecolone, Trichothecin, deoxynivalenol, 3-acetyldeoxynivalenol,5-acetyldeoxynivalenol, 3,15-diacetyldeoxynivalenol, Nivalenol,4-acetylnivalenol (Fusarenon-X), 4,15-idacetylnivalenol,4,7,15-triacetylnivalenol, and tetra-acetylnivalenol. Representativeexamples of Group C simple trichothecenes include: Crotocol andCrotocin. Representative macrocyclic trichothecenes include VerrucarinA, Verrucarin B, Verrucarin J (Satratoxin C), Roridin A, Roridin D,Roridin E (Satratoxin D), Roridin H, Satratoxin F, Satratoxin G,Satratoxin H, Vertisporin, Mytoxin A, Mytoxin C, Mytoxin B, Myrotoxin A,Myrotoxin B, Myrotoxin C, Myrotoxin D, Roritoxin A, Roritoxin B, andRoritoxin D. In addition, the general "trichothecene" sesquiterpenoidring structure is also present in compounds termed "baccharins" isolatedfrom the higher plant Baccharis megapotamica, and these are described inthe literature, for instance as disclosed by Jarvis et al. (Chemistry ofAlleopathy, ACS Symposium Series No. 268: ed. A. C. Thompson, 1984, pp.149-159).

Experimental drugs, such as mercaptopurine, N-methylformamide,2-amino-1,3,4-thiadiazole, melphalan, hexamethylmelamine, galliumnitrate, 3% thymidine, dichloromethotrexate, mitoguazone, suramin,bromodeoxyuridine, iododeoxyuridine, semustine,1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea,N,N'-hexamethylene-bis-acetamide, azacitidine, dibromodulcitol, Erwiniaasparaginase, ifosfamide, 2-mercaptoethane sulfonate, teniposide, taxol,3-deazauridine, soluble Baker's antifol, homoharringtonine,cyclocytidine, acivicin, ICRF-187, spiromustine, levamisole,chlorozotocin, aziridinyl benzoquinone, spirogermanium, aclarubicin,pentostatin, PALA, carboplatin, amsacrine, caracemide, iproplatin,misonidazole, dihydro-5-azacytidine, 4'-deoxy-doxorubicin, menogaril,triciribine phosphate, fazarabine, tiazofurin, teroxirone, ethiofos,N-(2-hydroxyethyl)-2-nitro-1H-imidazole-1-acetamide, mitoxantrone,acodazole, amonafide, fludarabine phosphate, pibenzimol, didemnin B,merbarone, dihydrolenperone, flavone-8-acetic acid, oxantrazole,ipomeanol, trimetrexate, deoxyspergualin, echinomycin, anddideoxycytidine (see NCI Investigational Drugs, Pharmaceutical Data1987, NIH Publication No. 88-2141, Revised November 1987) are alsopreferred.

Radionuclides useful within the present invention includegamma-emitters, positron-emitters, Auger electron-emitters, X-rayemitters and fluorescence-emitters, with beta- or alpha-emitterspreferred for therapeutic use. Radionuclides are well-known in the artand include ¹²³ I, ¹²⁵ I, ¹³⁰ I, ¹³¹ I, ¹³³ I, ¹³⁵ I, ⁴⁷ Sc, ⁷² As, ⁷²Se, ⁹⁰ Y, ⁸⁸ Y, ⁹⁷ Ru, ¹⁰⁰ Pd, ^(101m) Rh, ¹¹⁹ Sb, ¹²⁸ Ba, ¹⁹⁷ Hg, ²¹¹At, ²¹² Bi, ¹⁵³ Sm, ¹⁶⁹ Eu, ²¹² Pb, ¹⁰⁹ Pd, ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ⁶⁷ Cu,⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ^(99m) Tc, ¹¹ C, ¹³ N, ¹⁵ O and ¹⁸ F. Preferredtherapeutic radionuclides include ¹⁸⁸ Re, ¹⁸⁶ Re, ²⁰³ Pb, ²¹² Pb, ²¹²Bi, ¹⁰⁹ Pd, ⁶⁴ Cu, ⁶⁷ Cu, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ⁷⁷ Br, ²¹¹ At, ⁹⁷ Ru, ¹⁰⁵Rh, ¹⁹⁸ Au, ¹⁷⁷ Lu and ¹⁹⁹ Ag.

Other anti-tumor agents, e.g., agents active against proliferatingcells, are administrable in accordance with the present invention.Exemplary anti-tumor agents include cytokines, such as IL-2, tumornecrosis factor or the like, lectin inflammatory response promoters(selectins), such as L-selectin, E-selectin, P-selectin or the like, andlike molecules.

Ligands suitable for use within the present invention include biotin,haptens, lectins, epitopes, dsDNA fragments, enzyme inhibitors andanalogs and derivatives thereof. Useful complementary anti-ligandsinclude avidin (for biotin), carbohydrates (for lectins), antibody,fragments or analogs thereof, including mimetics (for haptens andepitopes), zinc finger proteins (for dsDNA fragments) and enzymes (forenzyme inhibitors. Preferred ligands and anti-ligands bind to each otherwith an affinity of at least about k_(D) ≧10⁻ 9M.

The 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetra acetic acid(DOTA)-biotin conjugate (DOTA-LC-biotin) depicted below has beenreported to have desirable in vivo biodistribution and is clearedprimarily by renal excretion. ##STR2## DOTA may also be conjugated toother ligands or to anti-ligands in the practice of the presentinvention.

Because DOTA strongly binds Y-90 and other radionuclides, it has beenproposed for use in radioimmunotherapy. For therapy, it is veryimportant that the radionuclide be stably bound within the DOTA chelateand that the DOTA chelate be stably attached to a ligand or anti-ligand.For illustrative purposes, DOTA-biotin conjugates are described. Onlyradiolabeled DOTA-biotin conjugates exhibiting those two characteristicsare useful to deliver radionuclides to the targets. Release of theradionuclide from the DOTA chelate or cleavage of the biotin and DOTAconjugate components in serum or at non-target sites renders theconjugate unsuitable for use in therapy.

Serum stability of DOTA-LC-biotin (where LC refers to the "long chain"linker, including an aminocaproyl spacer between the biotin and the DOTAconjugate components) shown above, while reported in the literature tobe good, has proven to be problematic. Experimentation has revealed thatDOTA-LC-biotin is rapidly cleared from the blood and excreted into theurine as fragments, wherein the biotinamide bond rather than theDOTA-amide bond has been cleaved, as shown below. ##STR3##

Additional experimentation employing PIP-biocytin conjugates producedparallel results as shown below. ##STR4## Cleavage of the benzamide wasnot observed as evidenced by the absence of detectable quantities ofiodobenzoic acid in the serum.

It appears that the cleavage results from the action of serumbiotinidase. Biotinidase is a hydrolytic enzyme that catalyzes thecleavage of blotin from biotinyl peptides. See, for example,Evangelatos, et al., "Biotinidase Radioassay Using an I-125-BiotinDerivative, Avidin, and Polyethylene Glycol Reagents," AnalyticalBiochemistry, 196: 385-89, 1991.

Drug-biotin conjugates which structurally resemble biotinyl peptides arepotential substrates for cleavage by plasma biotinidase. Poor in vivostability therefore limits the use of drug-biotin conjugates intherapeutic applications. The use of peptide surrogates to overcome poorstability of peptide therapeutic agents has been an area of intenseresearch effort. See, for example, Spatola, Peptide BackboneModification: A Structure-Activity Analysis of Peptide Containing AmideBond Surrogates, "Chemistry and Biochemistry of Amino Acids, Peptidesand Proteins," vol. 7, Weinstein, ed., Marcel Dekker, New York, 1983;and Kim et al., "A New Peptide Bond Surrogate: 2-Isoxazoline inPseudodipeptide Chemistry," Tetrahedron Letters, 45: 6811-14, 1991.

Elimination of the aminocaproyl spacer of DOTA-LC-biotin givesDOTA-SC-biotin (where the SC indicates the "short chain" linker betweenthe DOTA and biotin conjugate components), which molecule is shownbelow: ##STR5## DOTA-SC-biotin exhibits significantly improved serumstability in comparison to DOTA-LC-biotin. This result does not appearto be explainable on the basis of biotinidase activity alone. Theexperimentation leading to this conclusion is summarized in the Tableset forth below.

    ______________________________________                                        Time Dependent Cleavage of DOTA-Biotin conjugates                                       % Avidin Binding                                                                           Y-90-LC    Y-90-SC                                     Time at 37° C.                                                                     PIP-Biocytin                                                                             DOTA-Biotin                                                                              DOTA-Biotin                                 ______________________________________                                         5 Minutes  75%        50%        --                                          15 Minutes  57%        14%        --                                          30 Minutes  31%        12%        --                                          60 Minutes  --          0%        98%                                         20 Hours    --          0%        60%                                         ______________________________________                                         where "--" indicates that the value was not measured.                    

The difference in serum stability between DOTA-LC-biotin andDOTA-SC-biotin might be explained by the fact that the SC derivativecontains an aromatic amide linkage in contrast to the aliphatic amidelinkage of the LC derivative, with the aliphatic amide linkage beingmore readily recognized by enzymes as a substrate therefor. Thisargument cannot apply to biotinidase, however, because biotinidase veryefficiently cleaves aromatic amides. In fact, it is recognized that thesimplest and most commonly employed biotinidase activity measuringmethod uses N-(d-biotinyl)-4-aminobenzoate (BPABA) as a substrate, withthe hydrolysis of BPABA resulting in the liberation of biotin and4-aminobenzoate (PABA). See, for example, B. Wolf, et al., "Methods inEnzymology," pp. 103-111, Academic Press Inc., 1990. Consequently, onewould predict that DOTA-SC-biotin, like its LC counterpart, would be abiotinidase substrate. Since DOTA-SC-biotin exhibits serum stability,biotinidase activity alone does not adequately explain why someconjugates are serum stable while others are not, A series ofDOTA-biotin conjugates was therefore synthesized by the presentinventors to determine which structural features conferred serumstability to the conjugates.

Some general strategies for improving serum stability of peptides withrespect to enzymatic action are the following: incorporation of D-aminoacids, N-methyl amino acids and alpha-substituted amino acids.

In vivo stable biotin-DOTA conjugates are useful within the practice ofthe present invention. In vivo stability imparts the followingadvantages:

1) increased tumor uptake in that more of the radioisotope will betargeted to the previously localized targeting moiety-streptavidin; and2) increased tumor retention, if biotin is more stably bound to theradioisotope.

In addition, the linkage between DOTA and biotin may also have asignificant impact on biodistribution (including normal organ uptake,target uptake and the like) and pharmacokinetics.

The strategy for design of the DOTA-containing molecules and conjugatesof the present invention involved three primary considerations:

1) in vivo stability (including biotinidase and general peptidaseactivity resistance), with an initial acceptance criterion of 100%stability for 1 hour;

2) renal excretion; and

3) ease of synthesis.

The DOTA-biotin conjugates of the present invention reflect theimplementation of one or more of the following strategies:

1) substitution of the carbon adjacent to the cleavage susceptible amidenitrogen;

2) alkylation of the cleavage susceptible amide nitrogen;

3) substitution of the amide carbonyl with an alkyl amino group;

4) incorporation of D-amino acids as well as analogs or derivativesthereof; or

5) incorporation of thiourea linkages.

DOTA-biotin conjugates in accordance with the present invention may begenerally characterized as follows: conjugates that retain the biotincarboxy group in the structure thereof and those that do not (i.e., theterminal carboxy group of biotin has been reduced or otherwisechemically modified. Structures of such conjugates represented by thefollowing general formula have been devised: ##STR6## wherein L mayalternatively be substituted in one of the following ways on one of the--CH₂ --COOH branches of the DOTA structure: --CH(L)--COOH or --CH₂ COOLor --CH₂ COL). When these alternative structures are employed, theportion of the linker bearing the functional group for binding with theDOTA conjugate component is selected for the capability to interact witheither the carbon or the carboxy in the branch portions of the DOTAstructure, with the serum stability conferring portion of the linkerstructure being selected as described below.

In the case where the linkage is formed on the core of the DOTAstructure as shown above, L is selected according to the followingprinciples, with the portion of the linker designed to bind to the DOTAconjugate component selected for the capability to bind to an amine.

A. One embodiment of the present invention includes linkersincorporating a D-amino acid spacer between a DOTA aniline amine and thebiotin carboxy group shown above. Substituted amino acids are preferredfor these embodiments of the present invention, becausealpha-substitution also confers enzymatic cleavage resistance. ExemplaryL moieties of this embodiment of the present invention may berepresented as follows: ##STR7## where R¹ is selected from lower alkyl,lower alkyl substituted with hydrophilic groups (preferably, (CH₂)_(n)--OH, (CH₂)_(n) --OSO₃, (CH₂)_(n) --SO₃, ##STR8## where n is 1 or 2),glucuronide-substituted amino acids or other glucuronide derivatives;and

R² is selected from hydrogen, lower alkyl, substituted lower alkyl(e.g., hydroxy, sulfate, phosphonate or a hydrophilic moiety (preferablyOH).

For the purposes of the present disclosure, the term "lower alkyl"indicates an alkyl group with from one to five carbon atoms. Also, theterm "substituted" includes one or several substituent groups, with asingle substituent group preferred.

Preferred L groups of this embodiment of the present invention includethe following:

R¹ =CH₃ and R² =H (a D-alanine derivative, with a synthetic schemetherefor shown in Example XI); p1 R¹ =CH₃ and R² =CH₃ (anN-methyl-D-alanine derivative);

R¹ =CH₂ --OH and R² =H (a D-serine derivative);

R¹ =CH₂ OSO₃ and R² =H (a D-serine-O-sulfate-derivative); and ##STR9##and R² =H (a D-serine-O-phosphonate-derivative);

Other preferred moieties of this embodiment of the present inventioninclude molecules wherein R¹ is hydrogen and R² =--(CH₂)_(n) OH or asulfate or phosphonate derivative thereof and n is 1 or 2 as well asmolecules wherein R¹ is ##STR10##

Preferred moieties incorporating the glucuronide of D-lysine and theglucuronide of amino pimelate are shown below as I and II, respectively.##STR11##

A particularly preferred linker of this embodiment of the presentinvention is the D-alanine derivative set forth above.

B. Linkers incorporating alkyl substitution on one or more amidenitrogen atoms are also encompassed by the present invention, with someembodiments of such linkers preparable from L-amino acids. Amide bondshaving a substituted amine moiety are less susceptible to enzymaticcleavage. Such linkers exhibit the following general formula: ##STR12##where R⁴ is selected from hydrogen, lower alkyl, lower alkyl substitutedwith hydroxy, sulfate, phosphonate or the like and ##STR13##

R³ is selected from hydrogen; an amine; lower alkyl; an amino- or ahydroxy-, sulfate- or phosphonate-substituted lower alkyl; a glucuronideor a glucuronide-derivatized amino groups; and

n ranges from 0-4.

Preferred linkers of this embodiment of the present invention include=

R³ =H and R⁴ =CH₃ when n=4, synthesizable as discussed in Example XI;

R³ =H and R⁴ =CH₃ when n=0, synthesizable from N-methyl-glycine (havinga trivial name of sarcosine) as described in Example XI;

R³ =NH₂ and R⁴ =CH₃, when n=0;

R³ =H and ##STR14## when n=4 (Bis-DOTA-LC-blotin), synthesizablebromohexanolc acid as discussed in Example XI; and

R³ =H and ##STR15## when n=0 (bis-DOTA-SC-biotin), synthesizable fromiminodiacetic acid.

The synthesis of a conjugate including a linker wherein R³ is H and R⁴is --CH₂ CH₂ OH and n is 0 is also described in Example XI.Schematically, the synthesis of a conjugate of this embodiment of thepresent invention wherein n is 0, R³ is H and R⁴ is --CH₂ --COOH isshown below. ##STR16##

Bis-DOTA-LC-biotin, for example, offers the following advantages:

1) incorporation of two DOTA molecules on one biotin moiety increasesthe overall hydrophilicity of the biotin conjugate and thereby directsin vivo distribution to urinary excretion; and

2) substitution of the amide nitrogen adjacent to the biotin carboxylgroup blocks peptide and/or biotinidase cleavage at that site.

Bis-DOTA-LC-biotin, the glycine-based linker and the N-methylated linkerwhere R³ =H, R⁴ =CH₃, n=4 are particularly preferred linkers of thisembodiment of the present invention.

C. Another linker embodiment incorporates a thiourea moiety therein.Exemplary thiourea adducts of the present invention exhibit thefollowing general formula: ##STR17## where R⁵ is selected from hydrogenor lower alkyl;

R⁶ is selected from H and a hydrophilic moiety; and

n ranges from 0-4.

Preferred linkers of this embodiment of the present invention are asfollows:

R⁵ =H and R⁶ =H when n=5;

R⁵ =H and R⁶ =COOH when n=5; and

R⁵ =CH₃ and R⁶ =COOH when n=5.

The second preferred linker recited above can be prepared using eitherL-lysine or D-lysine. Similarly, the third preferred linker can beprepared using either N-methyl-D-lysine or N-methyl-L-lysine.

Another thiourea adduct of minimized lipophilicity is ##STR18## whichmay be formed via the addition of biotinhydrazide (commerciallyavailable from Sigma Chemical Co., St. Louis, Mo.) andDOTA-benzyl-isothiocyanate (a known compound synthesized in one stepfrom DOTA-aniline), with the thiourea-containing compound formed asshown below. ##STR19##

D. Amino acid-derived linkers of the present invention with substitutionof the carbon adjacent to the cleavage susceptible amide have thegeneral formula set forth below: ##STR20## wherein Z is --(CH₂)₂ --,conveniently synthesized form glutamic acid; or

Z=--CH₂ --S--CH₂ --, synthesizable from cysteine and iodo-acetic acid;or

Z=--CH₂ --, conveniently synthesized form aspartic acid; or

Z=--(CH₂)_(n) --CO--O--CH₂ --, where n ranges from 1-4 and which issynthesizable from serine.

E. Another exemplary linker embodiment of the present invention has thegeneral formula set forth below: ##STR21## and n ranges from 1-5.

F. Another embodiment involves disulfide-containing linkers, whichprovide a metabolically cleavable moiety (--S--S--) to reduce non-targetretention of the biotin-DOTA conjugate. Exemplary linkers of this typeexhibit the following formula: ##STR22## n and n' preferably rangebetween 0 and 5.

The advantage of using conditionally cleavable linkers is an improvementin target/non-target localization of the active agent. Conditionallycleavable linkers include enzymatically cleavable linkers, linkers thatare cleaved under acidic conditions, linkers that are cleaved underbasic conditions and the like. More specifically, use of linkers thatare cleaved by enzymes, which are present in non-target tissues butreduced in amount or absent in target tissue, can increase target cellretention of active agent relative to non-target cell retention. Suchconditionally cleavable linkers are useful, for example, in deliveringtherapeutic radionuclides to target cells, because such active agents donot require internalization for efficacy, provided that the linker isstable at the target cell surface or protected from target celldegradation.

Cleavable linkers are also useful to effect target site selectiverelease of active agent at target sites. Active agents that arepreferred for cleavable linker embodiments of the present invention arethose that are substantially non-cytotoxic when conjugated to ligand oranti-ligand. Such active agents therefore require release from theligand- or anti-ligand-containing conjugate to gain full potency. Forexample, such active agents, while conjugated, may be unable to bind toa cell surface receptor; unable to internalize either actively orpassively; or unable to serve as a binding substrate for a soluble(intra- or inter-cellular) binding protein or enzyme. Exemplary of anactive agent-containing conjugate of this type is chemotherapeuticdrug-cis-aconityl-biotin. The cis-aconityl linker is acid sensitive.Other acid sensitive linkers useful in cleavable linker embodiments ofthe present invention include esters, thioesters and the like. Use ofconjugates wherein an active agent and a ligand or an anti-ligand arejoined by a cleavable linker will result in the selective release of theactive agent at tumor cell target sites, for example, because theinter-cellular millieu of tumor tissue is generally of a lower pH (morehighly acidic) than the inter-cellular milieu of normal tissue.

G. Ether, thioether, ester and thioester linkers are also useful in thepractice of the present invention. Ether and thioether linkers arestable to acid and basic conditions and are therefore useful to deliveractive agents that are potent in conjugated form, such as radionuclidesand the like. Ester and thioesters are hydrolytically cleaved underacidic or basic conditions or are cleavable by enzymes includingesterases, and therefore facilitate improved target:non-targetretention. Exemplary linkers of this type have the following generalformula: ##STR23## X is O or S; and

Q is a bond, a methylene group, a --CO-- group or --CO--(CH₂)_(n)--NH--; and

n ranges from 1-5.

Other such linkers have the general formula:

--CH₂ --X--Q, where Q and X are defined as set forth above.

H. Another amino-containing linker of the present invention isstructured as follows: ##STR24## where R⁷ is lower alkyl, preferablymethyl. In this case, resistance to enzymatic cleavage is conferred bythe alkyl substitution on the amine.

I. Polymeric linkers are also contemplated by the present invention.Dextran and cyclodextran are preferred polymers useful in thisembodiment of the present invention as a result of the hydrophilicity ofthe polymer, which leads to favorable excretion of conjugates containingthe same. Other advantages of using dextran polymers are that suchpolymers are substantially non-toxic and non-immunogenic, that they arecommercially available in a variety of sizes and that they are easy toconjugate to other relevant molecules. Also, dextran-linked conjugatesexhibit advantages when non-target sites are accessible to dextranase,an enzyme capable of cleaving dextran polymers into smaller units whilenon-target sites are not so accessible.

Other linkers of the present invention are produced prior to conjugationto DOTA and following the reduction of the biotin carboxy moiety. Theselinkers of the present invention have the following general formula:##STR25##

Embodiments of linkers of this aspect of the present invention includethe following:

J. An ether linkage as shown below may be formed in a DOTA-biotinconjugate in accordance with the procedure indicated below.

    L'=--NH--CO--(CH.sub.2).sub.n --O--

where n ranges from 1 to 5, with 1 preferred. ##STR26## This linker hasonly one amide moiety which is bound directly to the DOTA aniline (as inthe structure of DOTA-SC-biotin). In addition, the ether linkage impartshydrophilicity, an important factor in facilitating renal excretion.

K. An amine linker formed from reduced biotin (hydroxybiotin oraminobiotin) is shown below, with conjugates containing such a linkerformed, for example, in accordance with the procedure described inExample XI.

    L'=--NH--

This linker contains no amide moieties and the unalkylated amine mayimpart favorable biodistribution properties since unalkylatedDOTA-aniline displays excellent renal clearance.

L. Substituted amine linkers, which can form conjugates via amino-biotinintermediates, are shown below. ##STR27## where R⁸ is H; --(CH₂)₂ --OHor a sulfate or phosphonate derivative thereof; or ##STR28## or thelike; and R⁹ is a bond or --(CH₂)_(n) --CO--NH--, where n ranges from0-5 and is preferably 1 and where q is 0 or 1. These moieties exhibitthe advantages of an amide only directly attached to DOTA-aniline andeither a non-amide amine imparting a positive charge to the linker invivo or a N-alkylated glucuronide hydrophilic group, each alternativefavoring renal excretion.

M. Amino biotin may also be used as an intermediate in the production ofconjugates linked by linkers having favorable properties, such as athiourea-containing linker of the formula:

L'=--NH--CS--NH--

Conjugates containing this thiourea linker have the followingadvantages: no cleavable amide and a short, fairly polar linker whichfavors renal excretion.

A bis-DOTA derivative of the following formula can also be formed fromamino-biotin. ##STR29## where n ranges from 1 to 5, with 1 and 5preferred. This molecule offers the advantages of the previouslydiscussed bis-DOTA derivatives with the added advantage of no cleavableamides.

Additional linkers of the present invention which are employed in theproduction of conjugates characterized by a reduced biotin carboxymoiety are the following:

L=--(CH₂)₄ --NH--, wherein the amine group is attached to the methylenegroup corresponding to the reduced biotin carboxy moiety and themethylene chain is attached to a core carbon in the DOTA ring. Such alinker is conveniently synthesizable from lysine.

L=--(CH₂)_(q) --CO--NH--, wherein q is 1 or 2, and wherein the aminegroup is attached to the methylene group corresponding to the reducedbiotin carboxy moiety and the methylene group(s) are attached to a corecarbon in the DOTA ring. This moiety is synthesizable from amino-biotin.

The linkers set forth above are useful to produce conjugates having oneor more of the following advantages:

bind avidin or streptavidin with the same or substantially similaraffinity as free biotin;

bind metal M⁺³ ions efficiently and with high kinetic stability;

are excreted primarily through the kidneys into urine;

are stable to bodily fluid amidases;

penetrate tissue rapidly and bind to pretargeted avidin or streptavidin;and

are excreted rapidly with a whole body residence half-life of less thanabout 5 hours.

Synthetic routes to an intermediate of the DOTA-biotin conjugatesdepicted above, nitrobenzyl-DOTA, have been proposed. These proposedsynthetic routes produce the intermediate compound in suboptimal yield,however. For example, Renn and Meares, "Large Scale Synthesis ofBifunctional Chelating AgentQ-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid, and the Determination of its Enantiomeric Purity by ChiralChromatography," Bioconj. Chem., 3: 563-9, 1992, describe a nine-stepsynthesis of nitrobenzyl-DOTA, including reaction steps that eitherproceed in low yield or involve cumbersome transformations orpurifications. More specifically, the sixth step proceeds in only 26%yield, and the product must be purified by preparative HPLC.Additionally, step eight proceeds in good yield, but the processinvolves copious volumes of the coreactants.

These difficulties in steps 6-8 of the prior art synthesis are overcomein the practice of the present invention through the use of thefollowing synthetic alternative therefor. ##STR30##

The poor yield in step six of the prior art synthesis procedure, inwhich a tetra amine alcohol is converted to a tetra-toluenesulfonamidetoluenesulfonate as shown below, is the likely result of prematureformation of the O-toluenesulfonate functionality (before all of theamine groups have been converted to their corresponding sulfonamides.##STR31## Such a sequence of events would potentially result in unwantedintra- or inter-molecular displacement of the reactiveO-toluenesulfonate by unprotected amine groups, thereby generatingnumerous undesirable side-products.

This problem is overcome in the aforementioned alternative synthesisscheme of the present invention by reacting the tetra-amine alcohol withtrifluoroacetic anhydride. Trifluoroacetates, being much poorer leavinggroups than toluenesulfonates, are not vulnerable to analogous sidereactions. In fact, the easy hydrolysis of trifluoroacetate groups, asreported in Greene and Wuts, "Protecting Groups in Organic Synthesis,"John Wiley and Sons, Inc., New York, p. 94, 1991, suggests that additionof methanol to the reaction mixture following consumption of all aminesshould afford the tetra-fluoroacetamide alcohol as a substantiallyexclusive product. Conversion of the tetra-fluoroacetamide alcohol tothe corresponding toluenesulfonate provides a material which is expectedto cyclize analogously to the tetra-toluenesulfonamide toluenesulfonateof the prior art. The cyclic tetraamide product of the cyclization ofthe toluenesulfonate of tetra-fluoroacetamide alcohol, in methanolicsodium hydroxide at 15°-25° C. for 1 hour, should affordnitro-benzyl-DOTA as a substantially exclusive product. As a result, theuse of trifluoracetamide protecting groups circumvents the difficultiesassociated with cleavage of the very stable toluenesulfonamideprotecting group, which involves heating with a large excess of sulfuricacid followed by neutralization with copious volumes of bariumhydroxide.

Another alternative route to nitro-benzyl-DOTA is shown below. ##STR32##This alternative procedure involves the cyclizaton ofp-nitrophenylalanyltriglycine using a coupling agent, such asdiethylycyanophosphate, to give the cyclic tetraamide. Subsequent boranereduction provides 2-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane, acommon precursor used in published routes to DOTA including the Renn andMeares article referenced above. This alternative procedure of thepresent invention offers a synthetic pathway that is considerablyshorter than the prior art Renn and Meares route, requiring two ratherthan four steps between p-nitrophenylalanyltriglycine to the tetraamine.The procedure of the present invention also avoids the use of tosylamino protecting groups, which were prepared in low yield and requiredstringent conditions for removal. Also, the procedure of the presentinvention poses advantages over the route published by Gansow et al.,U.S. Pat. No. 4,923,985, because the crucial cyclization step isintramolecular rather than intermolecular. Intramolecular reactionstypically proceed in higher yield and do not require high dilutiontechniques necessary for successful intermolecular reactions.

An additional aspect of the present invention is directed to the use oftargeting moieties that are monoclonal antibodies or fragments thereofthat localize to an antigen that is recognized by the antibody NR-LU-10.Such monoclonal antibodies or fragments may be murine or of othernon-human mammalian origin, chimeric, humanized or human.

NR-LU-10 is a 150 kilodalton molecular weight IgG2b monoclonal antibodythat recognizes an approximately 40 kilodalton glycoprotein antigenexpressed on most carcinomas. In vivo studies in mice using an antibodyspecific for the NR-LU-10 antigen revealed that such antibody was notrapidly internalized, which would have prevented localization of thesubsequently administered active-agent-containing conjugate to thetarget site.

NR-LU-10 is a well characterized pancarcinoma antibody that has beensafely administered to over 565 patients in human clinical trials. Thehybridoma secreting NR-LU-10 was developed by fusing mouse splenocytesimmunized with intact cells of a human small cell lung carcinoma withP3×63/Ag8UI murine myeloma cells. After establishing a seed lot, thehybridoma was grown via in vitro cell culture methods, purified andverified for purity and sterility.

Radioimmunoassays, immunoprecipitation and Fluorescence-Activated CellSorter (FACS) analysis were used to obtain reactivity profiles ofNR-LU-10. The NR-LU-10 target antigen was present on either fixedcultured cells or in detergent extracts of various types of cancercells. For example, the NR-LU-10 antigen is found in small cell lung,non-small cell lung, colon, breast, renal, ovarian, pancreatic, andother carcinoma tissues. Tumor reactivity of the NR-LU-10 antibody isset forth in Table A, while NR-LU-10 reactivity with normal tissues isset forth in Table B. The values in Table B are obtained as describedbelow. Positive NR-LU-10 tissue reactivity indicates NR-LU-10 antigenexpression by such tissues. The NR-LU-10 antigen has been furtherdescribed by Varki et al., "Antigens Associated with a Human LungAdenocarcinoma Defined by Monoclonal Antibodies," Cancer Research, 44:681-687, 1984, and Okabe et al., "Monoclonal Antibodies to SurfaceAntigens of Small Cell Carcinoma of the Lung," Cancer Research, 44:5273-5278, 1984.

The tissue specimens were scored in accordance with three reactivityparameters: (1) the intensity of the reaction; (2) the uniformity of thereaction within the cell type; and (3) the percentage of cells reactivewith the antibody. These three values are combined into a singleweighted comparative value between 0 and 500, with 500 being the mostintense reactivity. This comparative value facilitates comparison ofdifferent tissues. Table B includes a summary reactivity value, thenumber of tissue samples examined and the number of samples that reactedpositively with NR-LU-10.

Methods for preparing antibodies that bind to epitopes of the NR-LU-10antigen are described in U.S. Pat. No. 5,084,396. Briefly, suchantibodies may be prepared by the following procedure:

absorbing a first monoclonal antibody directed against a first epitopeof a polyvalent antigen onto an inert, insoluble matrix capable ofbinding immunoglobulin, thereby forming an immunosorbent;

combining the immunosorbent with an extract containing polyvalentNR-LU-10 antigen, forming an insolubilized immune complex wherein thefirst epitope is masked by the first monoclonal antibody;

                                      TABLE A                                     __________________________________________________________________________    TUMOR REACTIVITY OF ANTIBODY                                                  Organ/Cell Type                                                                             #Pos/                                                                             Intensity.sup.a                                                                      Percent.sup.b                                                                        Uniformity.sup.c                              Tumor         Exam                                                                              Avg. Range                                                                           Avg. Range                                                                           Avg. Range                                    __________________________________________________________________________    Pancreas Carcinoma                                                                          6/6 3  3   100                                                                              100 2.3                                                                              2-3                                        Prostate Carcinoma                                                                          9/9 2.8                                                                              2-3 95 80-100                                                                            2  1-3                                        Lung Adenocarcinoma                                                                         8/8 3  3   100                                                                              100 2.2                                                                              1-3                                        Lung Small Cell Carcinoma                                                                   2/2 3  3   100                                                                              100 2  2                                          Lung          8/8 2.3                                                                              2-3 73 5-100                                                                             1.8                                                                              1-3                                        Squamous Cell Carcinoma                                                       Renal Carcinoma                                                                             8/9 2.2                                                                              2-3 83 75-100                                                                            1  1                                          Breast Adenocarcinoma                                                                       23/23                                                                             2.9                                                                              2-3 97 75-100                                                                            2.8                                                                              1-3                                        Colon Carcinoma                                                                             12/12                                                                             2.9                                                                              2-3 98 95-100                                                                            2.9                                                                              2-3                                        Malignant Melanoma Ocular                                                                   0/2 0  0   0  0   0  0                                          Malignant Melanoma                                                                           0/11                                                                             0  0   0  0   0  0                                          Ovarian Carcinoma                                                                           35/35                                                                             2.9                                                                              2-3 200                                                                              100 2.2                                                                              1-3                                        Undifferentiated                                                                            1/1 2  2   90 90  2  2                                          Carcinoma                                                                     Osteasarcoma  1/1 2  2   20 20  1  1                                          Synovial Sarcoma                                                                            0/1 0  0   0  0   0  0                                          Lymphoma      0/2 0  0   0  0   0  0                                          Liposarcoma   0/2 0  0   0  0   0  0                                          Uterine Leiomyosarcoma                                                                      0/1 0  0   0  0   0  0                                          __________________________________________________________________________     .sup.a Rated from 0-3, with 3 representing highest intensity                  .sup.b Percentage of cells stained within the examined tissue section.        .sup.c Rates from 0-3, with 3 representing highest uniformity.           

                  TABLE B                                                         ______________________________________                                                                     Summary                                          Organ/Cell Type   # Pos/Exam Reactivity                                       ______________________________________                                        Adenoid                                                                       Epithelium        3/3        433                                              Lymphoid Follicle-Central                                                                       0/3        0                                                Lymphoid Follicle-Peripheral                                                                    0/3        0                                                Mucus Gland       2/2        400                                              Adipose Tissue                                                                Fat Cells         0/3        0                                                Adrenal                                                                       Zona Fasciculata Cortex                                                                         0/3        0                                                Zona Glomerulosa Cortex                                                                         0/3        0                                                Zona Reticularis Cortex                                                                         0/3        0                                                Medulla           0/3        0                                                Aorta                                                                         Endothelium       0/3        0                                                Elastic Interna   0/3        0                                                Tunica Adventitia 0/3        0                                                Tunica Media      0/3        0                                                Brain-Cerebellum                                                              Axons, Myelinated 0/3        0                                                Microglia         0/3        0                                                Neurons           0/3        0                                                Purkenje's Cells  0/3        0                                                Brain-Cerebrum                                                                Axons, Myelinated 0/3        0                                                Microglia         0/3        0                                                Neurons           0/3        0                                                Brain-Midbrain                                                                Axons, Myelinated 0/3        0                                                Microglia         0/3        0                                                Neurons           0/3        0                                                Colon                                                                         Mucosal Epithelium                                                                              3/3        500                                              Muscularis Externa                                                                              0/3        0                                                Muscularis Mucosa 0/3        0                                                Nerve Ganglia     0/3        0                                                Serosa            0/1        0                                                Duodenum                                                                      Mucosal Epithelium                                                                              3/3        500                                              Muscularis Mucosa 0/3        0                                                Epididymis                                                                    Epithelium        3/3        419                                              Smooth Muscle     0/3        0                                                Spermatozoa       0/1        0                                                Esophagus                                                                     Epithelium        3/3        86                                               Mucosal Gland     2/2        450                                              Smooth Muscle     0/3        0                                                Gall Bladder                                                                  Mucosal Epithelium                                                                              0/3        467                                              Smooth Muscle     0/3        0                                                Heart                                                                         Myocardium        0/3        0                                                Serosa            0/1        0                                                Ileum                                                                         Lymph Node        0/2        0                                                Mucosal Epithelium                                                                              0/2        0                                                Muscularis Externa                                                                              0/1        0                                                Muscularis Mucosa 0/2        0                                                Nerve Ganglia     0/1        0                                                Serosa            0/1        0                                                Jejunum                                                                       Lymph Node        0/1        0                                                Mucosal Epithelium                                                                              2/2        400                                              Muscularis Externa                                                                              0/2        0                                                Muscularis Mucosa 0/2        0                                                Nerve Ganglia     0/2        0                                                Serosa            0/1        0                                                Kidney                                                                        Collecting Tubules                                                                              2/3        160                                              Distal Convoluted Tubules                                                                       3/3        500                                              Glomerular Epithelium                                                                           0/3        0                                                Mesangial         0/3        0                                                Proximal Convoluted Tubules                                                                     3/3        500                                              Liver                                                                         Bile Duct         3/3        500                                              Central Lobular Hepatocyte                                                                      1/3        4                                                Periportal Hepatocyte                                                                           1/3        40                                               Kupffer Cells     0/3        0                                                Lung                                                                          Alveolar Macrophage                                                                             0/3        0                                                Bronchial Epithelium                                                                            0/2        0                                                Bronchial Smooth Muscle                                                                         0/2        0                                                Pneumocyte Type I 3/3        354                                              Pneumocyte Type II                                                                              3/3        387                                              Lymph Node                                                                    Lymphoid Follicle-Central                                                                       0/3        0                                                Lymphoid Follicle-Peripheral                                                                    0/3        0                                                Mammary Gland                                                                 Aveolar Epithelium                                                                              3/3        500                                              Duct Epithelium   3/3        500                                              Myoepithelium     0/3        0                                                Muscle Skeletal                                                               Muscle Fiber      0/3        0                                                Nerve                                                                         Axon, Myelinated  0/2        0                                                Endoneurium       0/2        0                                                Neurolemma        0/2        0                                                Neuron            0/2        0                                                Perineurium       0/2        0                                                Ovary                                                                         Corpus Luteum     0/3        0                                                Epithelium        1/1        270                                              Granulosa         1/3        400                                              Serosa            0/3        0                                                Theca             0/3        0                                                Oviduct                                                                       Epithelium        1/1        500                                              Smooth Muscle     0/3        0                                                Pancreas                                                                      Acinar Cell       3/3        500                                              Duct Epithelium   3/3        500                                              Islet Cell        3/3        500                                              Peritoneum                                                                    Mesothelium       0/1        0                                                Pituitary                                                                     Adenohypophysis   2/2        500                                              Neurohypophysis   0/2        0                                                Placenta                                                                      Trophoblasts      0/3        0                                                Prostate                                                                      Concretions       0/3        0                                                Glandular Epithelium                                                                            3/3        400                                              Smooth Muscle     0/3        0                                                Rectum                                                                        Lymph Node        0/2        0                                                Mucosal Epithelium                                                                              0/2        0                                                Muscularis Externa                                                                              0/1        0                                                Muscularis Mucosa 0/3        0                                                Nerve Ganglia     0/3        0                                                Salivary Gland                                                                Acinar Epithelium 3/3        500                                              Duct Epithelium   3/3        500                                              Skin                                                                          Apocrine Glands   3/3        280                                              Basal Layer       3/3        33                                               Epithelium        1/3        10                                               Follicle          1/1        190                                              Stratum Corneum   0/3        0                                                Spinal Cord                                                                   Axons, Myelinated 0/2        0                                                Microglial        0/2        0                                                Neurons           0/2        0                                                Spleen                                                                        Lymphoid Follicle-Central                                                                       0/3        0                                                Lymphoid Follicle-Peripheral                                                                    0/3        0                                                Trabecular Smooth Muscle                                                                        0/3        0                                                Stomach                                                                       Chief Cells       3/3        290                                              Mucosal Epithelium                                                                              3/3        367                                              Muscularis Mucosa/Externa                                                                       0/3        0                                                Parietal Cells    3/3        290                                              Smooth Muscle     0/3        0                                                Stromal Tissue                                                                Adipose            0/63      0                                                Arteriolar Smooth Muscle                                                                         0/120     0                                                Endothelium        0/120     0                                                Fibrous Connective Tissue                                                                        0/120     0                                                Macrophages        0/117     0                                                Mast Cells/Eosinophils                                                                           0/86      0                                                Testis                                                                        Interstitial Cells                                                                              0/3        0                                                Sertoli Cells     3/3        93                                               Thymus                                                                        Hassal's Epithelium                                                                             3/3        147                                              Hassal's Keratin  3/3        333                                              Lymphoid Cortex   0/3        0                                                Lymphold Medulla  3/3        167                                              Thyroid                                                                       C-cells           0/3        0                                                Colloid           0/3        0                                                Follicular Epithelium                                                                           3/3        500                                              Tonsil                                                                        Epithelium        1/3        500                                              Lymphoid Follicle-Central                                                                       0/3        0                                                Lymphoid Follicle-Peripheral                                                                    0/3        0                                                Mucus Gland       1/1        300                                              Striated Muscle   0/3        0                                                Umbilical cord                                                                Epithelium        0/3        0                                                Urinary Bladder                                                               Mucosal Epithelium                                                                              3/3        433                                              Serosa            0/1        0                                                Smooth Muscle     0/3        0                                                Uterus                                                                        Endometrial Epithelium                                                                          3/3        500                                              Endometrial Glands                                                                              3/3        500                                              Smooth Muscle     0/3        0                                                Vagina/Cervix                                                                 Epithelial Glands 1/1        500                                              Smooth Muscle     0/2        0                                                Squamous Epithelium                                                                             1/1        200                                              ______________________________________                                    

immunizing an animal with the insolubilized immune complex;

fusing spleen cells from the immunized animal to myeloma cells to form ahybridoma capable of producing a second monoclonal antibody directedagainst a second epitope of the polyvalent antigen;

culturing the hybridoma to produce the second monoclonal antibody; and

collecting the second monoclonal antibody as a product of the hybridoma.

Consequently, monoclonal antibodies NR-LU-01, NR-LU-02 and NR-LU-03,prepared in accordance with the procedures described in theaforementioned patent, are exemplary targeting moieties useful in thisaspect of the present invention.

Additional antibodies reactive with the NR-LU-10 antigen may also beprepared by standard hybridoma production and screening techniques. Anyhybridoma clones so produced and identified may be further screened asdescribed above to verify antigen and tissue reactivity.

The invention is further described through presentation of the followingexamples. These examples are offered by way of illustration, and not byway of limitation.

EXAMPLE I

Synthesis of a Chelate-Biotin Conjugate

A chelating compound that contains an N₃ S chelating core was attachedvia an amide linkage to biotin. Radiometal labeling of an exemplarychelate-biotin conjugate is illustrated below. ##STR33##

The spacer group "X" permits the biotin portion of the conjugate to besterically available for avidin binding. When "R¹ " is a carboxylic acidsubstituent (for instance, CH₂ COOH), the conjugate exhibits improvedwater solubility, and further directs in vivo excretion of theradiolabeled biotin conjugate toward renal rather than hepatobiliaryclearance.

Briefly, N-α-Cbz-N-Σ-t-BOC protected lysine was converted to thesuccinimidyl ester with NHS and DCC, and then condensed with asparticacid β-t-butyl ester. The resultant dipeptide was activated with NHS andDCC, and then condensed With glycine t-butyl ester. The Cbz group wasremoved by hydrogenolysis, and the amine was acylated usingtetrahydropyranyl mercaptoacetic acid succinimidyl ester, yieldingS-(tetrahydropyranyl)-mercaptoacetyl-lysine. Trifluoroacetic acidcleavage of the N-t-BOC group and t-butyl esters, followed bycondensation with LC-biotin-NHS ester provided (Σ-caproylamidebiotin)aspartyl glycine. This synthetic method is illustrated below.##STR34##

¹ H NMR: (CD₃ OD, 200 MHz Varian): 1.25-1.95 (m, 24H), 2.15-2.25 (broadt, 4H), 2.65-3.05 (m, 4H), 3.30-3.45 (dd, 2H), 3.50-3.65 (ddd, 2H), 3.95(broad s, 2H), 4.00-4.15 (m, 1H), 4.25-4.35 (m, 1H), 4.45-4.55 (m, 1H),4.7-5.05 (m overlapping with HOD).

Elemental Analysis: C, H, N for C₃₅ H₅₇ N₇ O₁₁ S₂ ·H₂ O; calculated:50.41, 7.13, 11.76; found: 50.13, 7.14, 11.40.

EXAMPLE II

Preparation of a Technetium or Rhenium Radiolabeled Chelate-BiotinConjugate

The chelate-biotin conjugate of Example I was radiolabeled with either^(99m) Tc pertechnetate or ¹⁸⁶ Re perrhenate. Briefly, ^(99m) Tcpertechnetate was reduced with stannous chloride in the presence ofsodium gluconate to form an intermediate Tc-gluconate complex. Thechelate-biotin conjugate of Example I was added and heated to 100° C.for 10 min at a pH of about 1.8 to about 3.3. The solution wasneutralized to a pH of about 6 to about 8, and yielded an N₃S-coordinated ^(99m) Tc-chelate-biotin conjugate. C-18 HPLC gradientelution using 5-60% acetonitrile in 1% .acetic acid demonstrated twoanomers at 97% or greater radiochemical yield using δ detection.

Alternatively, ¹⁸⁶ Re perrhenate was spiked with cold ammoniumperrhenate, reduced with stannous chloride, and complexed with citrate.The chelate-biotin conjugate of Example I was added and heated to 90° C.for 30 min at a pH of about 2 to 3. The solution was neutralized to a pHof about 6 to about 8, and yielded an N₃ S-coordinated ¹⁸⁶Re-chelate-biotin conjugate. C-18 HPLC gradient elution using 5-60%acetonitrile in 1% acetic acid resulted in radiochemical yields of85-90%. Subsequent purification over a C-18 reverse phase hydrophobiccolumn yielded material of 99% purity.

EXAMPLE III

In Vitro Analysis of Radiolabeled Chelate-Biotin Conjugates

Both the ^(99m) Tc- and ¹⁸⁶ Re-chelate-biotin conjugates were evaluatedin vitro. When combined with excess avidin (about 100-fold molarexcess), 100% of both radiolabeled biotin conjugates complexed withavidin.

A ^(99m) Tc-biotin conjugate was subjected to various chemical challengeconditions. Briefly, ^(99m) Tc-chelate-biotin conjugates were combinedwith avidin and passed over a 5 cm size exclusion gel filtration column.The radiolabeled biotin-avidin complexes were subjected to variouschemical challenges (see Table 1), and the incubation mixtures werecentrifuged through a size exclusion filter. The percent ofradioactivity retained (indicating avidin-biotin-associated radiolabel)is presented in Table 1. Thus, upon chemical challenge, the radiometalremained associated with the macromolecular complex.

                  TABLE 1                                                         ______________________________________                                        Chemical Challenge of .sup.99m Tc-Chelate-                                    Biotin-Avidin Complexes                                                       Challenge              % Radioactivity Retained                               Medium      pH         1 h, 37° C.                                                                      18 h, RT                                     ______________________________________                                        PBS         7.2        99        99                                           Phosphate   8.0        97        97                                           10 mM cysteine                                                                            8.0        92        95                                           10 mM DTPA  8.0        99        98                                           0.2 M carbonate                                                                           10.0       97        94                                           ______________________________________                                    

In addition, each radiolabeled biotin conjugate was incubated at about50 μg/ml with serum; upon completion of the incubation, the samples weresubjected to instant thin layer chromatography (ITLC) in 80% methanol.Only 2-4% of the radioactivity remained at the origin (i.e., associatedwith protein); this percentage was unaffected by the addition ofexogenous biotin. When the samples were analyzed using size exclusionH-12 FPLC with 0.2M phosphate as mobile phase, no association ofradioactivity with serum macromolecules was observed.

Each radiolabeled biotin conjugate was further examined using acompetitive biotin binding assay. Briefly, solutions containing varyingratios of D-biotin to radiolabeled biotin conjugate were combined withlimiting avidin at a constant total biotin:avidin radio. Avidin bindingof each radiolabeled biotin conjugate was determined by ITLC, and wascompared to the theoretical maximum stoichiometric binding (asdetermined by the HABA spectrophotometric assay of Green, Biochem. J.94:23c-24c, 1965). No significant difference in avidin binding wasobserved between each radiolabeled biotin conjugate and D-biotin.

EXAMPLE IV

In Vivo Analysis of Radiolabeled Chelate-Biotin Conjugates AdministeredAfter Antibody Pretargeting

The ¹⁸⁶ Re-chelate-biotin conjugate of Example I was studied in ananimal model of a three-step antibody pretargeting protocol. Generally,this protocol involved: (i) prelocalization of biotinylated monoclonalantibody; (ii) administration of avidin for formation of a "sandwich" atthe target site and for clearance of residual circulating biotinylatedantibody; and (iii) administration of the 186Re-biotin conjugate fortarget site localization and rapid blood clearance.

A. Preparation and Characterization of Biotinylated Antibody

Biotinylated NR-LU-10 was prepared according to either of the followingprocedures. The first procedure involved derivatization of antibody vialysine ε-amino groups. NR-LU-10 was radioiodinated at tyrosines usingchloramine T and either ¹²⁵ I or ¹³¹ I sodium iodide. The radioiodinatedantibody (5-10 mg/ml) was then biotinylated using biotinamido caproateNHS ester in carbonate buffer, pH 8.5, containing 5% DMSO, according tothe scheme below. ##STR35##

The impact of lysine biotinylation on antibody immunoreactivity wasexamined. As the molar offering of biotin:antibody increased from 5:1 to40:1, biotin incorporation increased as expected (measured using theHABA assay and pronase-digested product) (Table 2, below). Percent ofbiotinylated antibody immunoreactivity as compared to native antibodywas assessed in a limiting antigen ELISA assay. The immunoreactivitypercentage dropped below 70% at a measured derivitization of 11.1:1;however, at this level of derivitization, no decrease inantigen-positive cell binding (performed with LS-180 tumor cells atantigen excess). Subsequent experiments used antibody derivitized at abiotin:antibody ratio of 10:1.

                  TABLE 2                                                         ______________________________________                                        Effect of Lysine Biotinylation                                                on Immunoreactivity                                                           Molar      Measured                                                           Offering   Derivitization                                                                             Immunoassessment (%)                                  (Biotins/Ab)                                                                             (Biotins/Ab) ELISA   Cell Binding                                  ______________________________________                                         5:1        3.4         86                                                    10:1        8.5         73      100                                           13:1       11.1         69      102                                           20:1       13.4         36      106                                           40:1       23.1         27                                                    ______________________________________                                    

Alternatively, NR-LU-10 was biotinylated using thiol groups generated byreduction of cystines. Derivitization of thiol groups was hypothesizedto be less compromising to antibody immunoreactivity. NR-LU-10 wasradioiodinated using p-aryltin phenylate NHS ester (PIP-NHS) and either¹²⁵ I or ¹³¹ I sodium iodide. Radioiodinated NR-LU-10 was incubated with25 mM dithiothreitol and purified using size exclusion chromatography.The reduced antibody (containing free thiol groups) was then reactedwith a 10- to 100-fold molar excess of N-iodoacetyl-n'-biotinyl hexylenediamine in phosphate-buffered saline (PBS), pH 7.5, containing 5% DMSO(v/v).

                  TABLE 3                                                         ______________________________________                                        Effect of Thiol Biotinylation                                                 on Immunoreactivity                                                           Molar      Measured                                                           Offering   Derivitization                                                                             Immunoassessment (%)                                  (Biotins/Ab)                                                                             (Biotins/Ab) ELISA   Cell Binding                                  ______________________________________                                        10:1       4.7          114                                                   50:1       6.5          102     100                                           100:1      6.1           95     100                                           ______________________________________                                    

As shown in Table 3, at a 50:1 or greater biotin:antibody molaroffering, only 6 biotins per antibody were incorporated. No significantimpact on immunoreactivity was observed.

The lysine- and thiol-derivitized biotinylated antibodies ("antibody(lysine)" and "antibody (thiol)", respectively) were compared. Molecularsizing on size exclusion FPLC demonstrated that both biotinylationprotocols yielded monomolecular IgGs. Biotinylated antibody (lysine) hadan apparent molecular weight of 160 kD, while biotinylated antibody(thiol) had an apparent molecular weight of 180 kD. Reduction ofendogenous sulfhydryls (i.e., disulfides) to thiol groups, followed byconjugation with biotin, may produce a somewhat unfolded macromolecule.If so, the antibody (thiol) may display a larger hydrodynamic radius andexhibit an apparent increase in molecular weight by chromatographicanalysis. Both biotinylated antibody species exhibited 98% specificbinding to immobilized avidin-agarose.

Further comparison of the biotinylated antibody species was performedusing non-reducing SDS-PAGE, using a 4% stacking gel and a 5% resolvinggel. Biotinylated samples were either radiolabeled or unlabeled and werecombined with either radiolabeled or unlabeled avidin or streptavidin.Samples were not boiled prior to SDS-PAGE analysis. The native antibodyand biotinylated antibody (lysine) showed similar migrations; thebiotinylated antibody (thiol) produced two species in the 50-75 kDrange. These species may represent two thiol-capped species. Under theseSDS-PAGE conditions, radiolabeled streptavidin migrates as a 60 kDtetramer. When 400 μg/ml radiolabeled streptavidin was combined with 50μg/ml biotinylated antibody (analogous to "sandwiching" conditions invivo), both antibody species formed large molecular weight complexes.However, only the biotinylated antibody (thiol)-streptavidin complexmoved from the stacking gel into the resolving gel, indicating adecreased molecular weight as compared to the biotinylated antibody(lysine ) -streptavidin complex.

B. Blood Clearance of Biotinylated Antibody Species

Radioiodinated biotinylated NR-LU-10 (lysine or thiol) was intravenouslyadministered to non-tumored nude mice at a dose of 100 μg. At 24 hpost-administration of radioiodinated biotinylated NR-LU-10, mice wereintravenously injected with either saline or 400 μg of avidin. Withsaline administration, blood clearances for both biotinylated antibodyspecies were biphasic and similar to the clearance of native NR-LU-10antibody.

In the animals that received avidin intravenously at 24 h, thebiotinylated antibody (lysine) was cleared (to a level of 5% of injecteddose) within 15 min of avidin administration (avidin:biotin=10:1). Withthe biotinylated antibody (thiol), avidin administration (10:1 or 25:1)reduced the circulating antibody level to about 35% of injected doseafter two hours. Residual radiolabeled antibody activity in thecirculation after avidin administration was examined in vitro usingimmobilized biotin. This analysis revealed that 85% of the biotinylatedantibody was complexed with avidin. These data suggest that thebiotinylated antibody (thiol)-avidin complexes that were formed wereinsufficiently crosslinked to be cleared by the RES.

Blood clearance and biodistribution studies of biotinylated antibody(lysine) 2 h post-avidin or post-saline administration were performed.Avidin administration significantly reduced the level of biotinylatedantibody in the blood (see FIG. 1), and increased the level ofbiotinylated antibody in the liver and spleen. Kidney levels ofbiotinylated antibody were similar.

EXAMPLE V

In Vivo Characterization of ¹⁸⁶ Re-Chelate-Biotin Conjugates in aThree-Step Pretargeting Protocol

A ¹⁸⁶ Re-chelate-biotin conjugate of Example I (MW≈1000; specificactivity=1-2 mCi/mg) was examined in a three-step pretargeting protocolin an animal model. More specifically, 18-22 g female nude mice wereimplanted subcutaneously with LS-180 human colon tumor xenografts,yielding 100-200 mg tumors within 10 days of implantation.

NR-LU-10 antibody (MW≈150 kD) was radiolabeled with ¹²⁵ I/Chloramine Tand biotinylated via lysine residues (as described in Example VI.A,above). Avidin (MW≈66 kD) was radiolabeled with ¹³¹ I/PIP-NHS (asdescribed for radioiodination of NR-LU-10 in Example IV.A., above). Theexperimental protocol was as follows:

    ______________________________________                                        Group 1:    Time 0, inject 100 μg .sup.125 I-labeled,                                  biotinylated NR-LU-10                                                         Time 24 h, inject 400 μg .sup.131 I-labeled                                avidin                                                                        Time 26 h, inject 60 μg .sup.186 Re-chelate-                               biotin conjugate                                                  Group 2:    Time 0, inject 400 μg .sup.131 I-labeled avidin                (control)   Time 2 h, inject 60 μg .sup.186 Re-chelate-                                biotin conjugate                                                  Group 3:    Time 0, inject 60 μg .sup.186 Re-chelate-                      (control)   biotin conjugate                                                  ______________________________________                                    

The three radiolabels employed in this protocol are capable of detectionin the presence of each other. It is also noteworthy that the sizes ofthe three elements involved are logarithmicallydifferent--antibody≅150,000; avidin≅66,000; and biotin≅1,000.Biodistribution analyses were performed at 2, 6, 24, 72 and 120 h afteradministration of the ¹⁸⁶ Re-chelate-biotin conjugate.

Certain preliminary studies were performed in the animal model prior toanalyzing the ¹⁸⁶ Re-chelate-biotin conjugate in a three-steppretargeting protocol. First, the effect of biotinylated antibody onblood clearance of avidin was examined. These experiments showed thatthe rate and extent of avidin clearance was similar in the presence orabsence of biotinylated antibody. Second, the effect of biotinylatedantibody and avidin on blood clearance of the ¹⁸⁶ Re-chelate-biotinconjugate was examined; blood clearance was similar in the presence orabsence of biotinylated antibody and avidin.

Third, tumor uptake of biotinylated antibody administered at time 0 orof avidin administered at time 24 h was examined. At 25 h, about 350pmol/g biotinylated antibody was present at the tumor; at 32 h the levelwas about 300 pmol/g; at 48 h, about 200 pmol/g; and at 120 h, about 100pmol/g. Avidin uptake at the same time points was about 250, 150, 50 and0 pmol/g, respectively. From the same experiment, tumor to blood ratioswere determined for biotinylated antibody and for avidin. From 32 h to120 h, the ratios of tumor to blood were very similar.

The three-step pretargeting protocol (described for Group 1, above) wasthen examined. More specifically, tumor uptake of the ¹⁸⁶Re-chelate-biotin conjugate in the presence or absence of biotinylatedantibody and avidin was determined. In the absence of biotinylatedantibody and avidin, the ¹⁸⁶ Re-chelate-biotin conjugate displayed aslight peak 2 h post-injection, which was substantially cleared from thetumor by about 5 h. In contrast, at 2 h post-injection in the presenceof biotinylated antibody and avidin (specific), the ¹⁸⁶Re-chelate-biotin conjugate reached a peak in tumor approximately 7times greater than that observed in the absence of biotinylated antibodyand avidin. Further, the specifically bound ¹⁸⁶ Re-chelate-biotinconjugate was retained at the tumor at significant levels for more than50 h. Tumor to blood ratios determined in the same experiment increasedsignificantly over time (i.e., T:B=≈8 at 30 h; ≈15 at 100 h; ≈35 at 140h).

Tumor uptake of the ¹⁸⁶ Re-chelate-biotin conjugate has further beenshown to be dependent on the dose of biotinylated antibody administered.At 0 μg of biotinylated antibody, about 200 pmol/g of ¹⁸⁶Re-chelate-biotin conjugate was present at the tumor at 2 h afteradministration; at 50 μg antibody, about 500 pmol/g of ¹⁸⁶Re-chelate-biotin conjugate; and at 100 μg antibody, about 1,300 pmol/gof ¹⁸⁶ Re-chelate-biotin conjugate.

Rhenium tumor uptake via the three-step pretargeting protocol wascompared to tumor uptake of the same antibody radiolabeled throughchelate covalently attached to the antibody (conventional procedure).The results of this comparison are depicted in FIG. 2. Blood clearanceand tumor uptake were compared for the chelate directly labeled rheniumantibody conjugate and for the three-step pretargeted sandwich. Areasunder the curves (AUC) and the ratio of AUC_(tumor) /AUC_(blood) weredetermined. For the chelate directly labeled rhenium antibody conjugate,the ratio of AUC_(tumor) /AUC_(blood) =24055/10235; for the three-steppretargeted sandwich, the ratio of AUC_(tumor) /AUC_(blood) =46764/6555.

EXAMPLE VI

Preparation of Chelate-Biotin Conjugates Having Improved BiodistributionProperties

The biodistribution of ¹¹¹ In-labeled-biotin derivatives varies greatlywith structural changes in the chelate and the conjugating group.Similar structural changes may affect the biodistribution of technetium-and rhenium-biotin conjugates. Accordingly, methods for preparingtechnetium- and rhenium-biotin conjugates having optimal clearance fromnormal tissue are advantageous.

A. Neutral MAMA Chelate/Conjugate

A neutral MAMA chelate-biotin conjugate is prepared according to thefollowing scheme.

a) MAMA ligand ##STR36## The resultant chelate-biotin conjugate showssuperior kidney excretion. Although the net overall charge of theconjugate is neutral, the polycarboxylate nature of the moleculegenerates regions of hydrophilicity and hydrophobicity. By altering thenumber and nature of the carboxylate groups within the conjugate,excretion may be shifted from kidney to gastrointestinal routes. Forinstance, neutral compounds are cleared by the kidneys; anioniccompounds are cleared through the GI system.

B. Polylysine Derivitization

Conjugates containing polylysine may also exhibit beneficialbiodistribution properties. With whole antibodies, derivitization withpolylysine may skew the biodistribution of conjugate toward liveruptake. In contrast, derivitization of Fab fragments with polylysineresults in low levels of both liver and kidney uptake; blood clearanceof these conjugates is similar to that of Fab covalently linked tochelate. An exemplary polylysine derivitized chelate-biotin conjugate isillustrated below. ##STR37## Inclusion of polylysine inradiometal-chelate-biotin conjugates is therefore useful for minimizingor eliminating RES sequestration while maintaining good liver and kidneyclearance of the conjugate. Polylysine derivatives offer the furtheradvantages of: (1) increasing the specific activity of theradiometal-chelate-biotin conjugate; (2) permitting control of rate androute of blood clearance by varying the molecular weight of thepolylysine polymer; and (3) increasing the circulation half-life of theconjugate for optimal tumor interaction.

Polylysine derivitization is accomplished by standard methodologies.Briefly, poly-L-lysine is acylated according to standard amino groupacylation procedures (aqueous bicarbonate buffer, pH 8, added biotin-NHSester, followed by chelate NHS ester). Alternative methodology involvesanhydrous conditions using nitrophenyl esters in DMSO and triethylamine. The resultant conjugates are characterized by UV and NMR specta.

The number of biotins attached to polylysine is determined by the HABAassay. Spectrophotometric titration is used to assess the extent ofamino group derivitization. The radiometal-chelate-biotin conjugate ischaracterized by size exclusion.

C. Cleavable Linkage

Through insertion of a cleavable linker between the chelate and biotinportion of a radiometal-chelate-biotin conjugate, retention of theconjugate at the tumor relative to normal tissue may be enhanced. Morespecifically, linkers that are cleaved by enzymes present in normaltissue but deficient or absent in tumor tissue can increase tumorretention. As an example, the kidney has high levels of γ-glutamyltransferase; other normal tissues exhibit in vivo cleavage of γ-glutamylprodrugs. In contrast, tumors are generally deficient in enzymepeptidases. The glutamyl-linked biotin conjugate depicted below iscleaved in normal tissue and retained in the tumor. ##STR38##

D. Serine Linker With O-Polar Substituent

Sugar substitution of N₃ S chelates renders such chelates water soluble.Sulfonates, which are fully ionized at physiological pH, improve watersolubility of the chelate-biotin conjugate depicted below. ##STR39##This compound is synthesized according to the standard reactionprocedures. Briefly, biocytin is condensed with N-t-BOC-(O-sulfonate orO-glucose) serine NHS ester to give N-t-BOC-(O-sulfonate or O-glucose)serine biocytinamide. Subsequent cleavage of the N-t-BOC group with TFAand condensation with ligand NHS ester in DMF with triethylamineprovides ligand-amidoserine(O-sulfonate or O-glucose)biocytinamide.

EXAMPLE VII

Preparation and Characterization of PIP-Radioiodinated Biotin

Radioiodinated biotin derivatives prepared by exposure of poly-L-lysineto excess NHS-LC-biotin and then to Bolton-Hunter N-hydroxysuccinimideesters in DMSO has been reported. After purification, this product wasradiolabeled by the iodogen method (see, for instance, Del Rosario etal., J. Nucl. Med. 32:5, 1991, 993 (abstr.)). Because of the highmolecular weight of the resultant radioiodinated biotin derivative, onlylimited characterization of product (i.e., radio-HPLC and binding toimmobilized streptavidin) was possible.

Preparation of radioiodonated biotin according to the present inventionprovides certain advantages. First, the radioiodobiotin derivative is alow molecular weight compound that is amenable to complete chemicalcharacterization. Second, the disclosed methods for preparation involvea single step and eliminate the need for a purification step.

Briefly, iodobenzamide derivatives corresponding to biocytin (R=COOH)and biotinamidopentylamine (R=H) were prepared according to thefollowing scheme. In this scheme, "X" may be any radiohalogen, including¹²⁵ I, ¹³¹ I, ¹²³ I, ²¹¹ At and the like. ##STR40## Preparation of 1 wasgenerally according to Wilbur et al., J. Nucl. Med. 30:216-26, 1989,using a tributyltin intermediate. Water soluble carbodiimide was used inthe above-depicted reaction, since the NHS ester 1 formed intractablemixtures with DCU. The NHS ester was not compatible with chromatography;it was insoluble in organic and aqueous solvents and did not react withbiocytin in DMF or in buffered aqueous acetonitrile. The reactionbetween 1 and biocytin or 5-(biotinamido) pentylamine was sensitive tobase. When the reaction of 1 and biocytin or the pentylamine wasperformed in the presence of triethylamine in hot DMSO, formation ofmore than one biotinylated product resulted. In contrast, the reactionwas extremely clean and complete when a suspension of 1 and biocytin (4mg/ml) or the pentylamine (4 mg/ml) was heated in DMSO at 117° C. forabout 5 to about 10 min. The resultant ¹²⁵ I-biotin derivatives wereobtained in 94% radiochemical yield. Optionally, the radioiodinatedproducts may be purified using C-18 HPLC and a reverse phase hydrophobiccolumn. Hereinafter, the resultant radioiodinated products 2 arereferred to as PIP-biocytin (R=COOH) and PIP-pentylamine (R=H).

Both iodobiotin derivatives 2 exhibited ≧95% binding to immobilizedavidin. Incubation of the products 2 with mouse serum resulted in noloss of the ability of 2 to bind to immobilized avidin. Biodistributionstudies of 2 in male BALB/c mice showed rapid clearance from the blood(similar to ¹⁸⁶ Re-chelate-biotin conjugates described above). Theradioiodbiotin 2 had decreased hepatobiliary excretion as compared tothe ¹⁸⁶ Re-chelate-biotin conjugate; urinary excretion was increased ascompared to the ¹⁸⁶ Re-chelate-biotin conjugate. Analysis of urinarymetabolites of 2 indicated deiodination and cleavage of the biotin amidebond; the metabolites showed no binding to immobilized avidin. Incontrast, metabolites of the ¹⁸⁶ Re-chelate-biotin conjugate appear tobe excreted in urine as intact biotin conjugates. Intestinal uptake of 2is <50% that of the ¹⁸⁶ Re-chelate-biotin conjugate. Thesebiodistribution properties of 2 provided enhanced whole body clearanceof radioisotope and indicate the advantageous use of 2 withinpretargeting protocols.

¹³¹ I-PIP-biocytin was evaluated in a two-step pretargeting procedure intumor-bearing mice. Briefly, female nude mice were injectedsubcutaneously with LS-180 tumor cells; after 7 d, the mice displayed50-100 mg tumor xenografts. At t=0, the mice were injected with 200 μgof NR-LU-10-avidin conjugate labeled with ¹²⁵ I using PIP-NHS (seeExample IV.A.). At t=36 h, the mice received 42 μg of ¹³¹I-PIP-biocytin. The data showed immediate, specific tumor localization,corresponding to ≈1.5 ¹³¹ I-PIP-biocytin molecules per avidin molecule.

The described radiohalogenated biotin compounds are amenable to the sametypes of modifications described in Example VI above for ¹⁸⁶Re-chelate-biotin conjugates. In particular, the followingPIP-polylysine-biotin molecule is made by trace labeling polylysine with¹²⁵ I-PIP, followed by extensive biotinylation of the polylysine.##STR41## Assessment of ¹²⁵ I binding to immobilized avidin ensures thatall radioiodinated species also contain at least an equivalent ofbiotin.

EXAMPLE VIII

Preparation of Biotinylated Antibody (Thiol) Through Endogenous AntibodySulfhydryl Groups or Sulfhydryl-Generating Compounds

Certain antibodies have available for reaction endogenous sulfhydrylgroups. If the antibody to be biotinylated contains endogenoussulfhydryl groups, such antibody is reacted withN-iodoacetyl-n'-biotinyl hexylene diamine (as described in ExampleIV.A., above). The availability of one or more endogenous sulfhydrylgroups obviates the need to expose the antibody to a reducing agent,such as DTT, which can have other detrimental effects on thebiotinylated antibody.

Alternatively, one or more sulfhydryl groups are attached to a targetingmoiety through the use of chemical compounds or linkers that contain aterminal sulfhydryl group. An exemplary compound for this purpose isiminothiolane. As with endogenous sulfhydryl groups (discussed above),the detrimental effects of reducing agents on antibody are therebyavoided.

EXAMPLE IX

Two-Step Pretargeting Methodology That Does Not Induce Internalization

A NR-LU-13-avidin conjugate is prepared as follows. Initially, avidin isderivatized with N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). SMCC-derivedavidin is then incubated with NR-LU-13 in a 1:1 molar ratio at pH 8.5for 16 h. Unreacted NR-LU-13 and SMCC-derived avidin are removed fromthe mixture using preparative size exclusion HPLC. Two conjugates areobtained as products--the desired 1:1 NR-LU-13-avidin conjugate as themajor product; and an incompletely characterized component as the minorproduct.

A ^(99m) Tc-chelate-biotin conjugate is prepared as in Example II,above. The NR-LU-13-avidin conjugate is administered to a recipient andallowed to clear from the circulation. One of ordinary skill in the artof radioimmunoscintigraphy is readily able to determine the optimal timefor NR-LU-13-avidin conjugate tumor localization and clearance from thecirculation. At such time, the ^(99m) Tc-chelate-biotin conjugate isadministered to the recipient. Because the 99mTc-chelate-biotinconjugate has a molecular weight of ≈1,000, crosslinking ofNR-LU-13-avidin molecules on the surface of the tumor cells isdramatically reduced or eliminated. As a result, the ^(99m) Tcdiagnostic agent is retained at the tumor cell surface for an extendedperiod of time. Accordingly, detection of the diagnostic agent byimaging techniques is optimized; further, a lower dose of radioisotopeprovides an image comparable to that resulting from the typicalthree-step pretargeting protocol.

Optionally, clearance of NR-LU-13-avidin from the circulation may beaccelerated by plasmapheresis in combination with a biotin affinitycolumn. Through use of such column, circulating NR-LU-13-avidin will beretained extracaporeally, and the recipient's immune system exposure toa large, proteinaceous immunogen (i.e., avidin) is minimized.

An alternative procedure for clearing NR-LU-13-avidin from thecirculation without induction of internalization involves administrationof biotinylated, high molecular weight molecules, such as liposomes, IgMand other molecules that are size excluded from ready permeability totumor sites. When such biotinylated, high molecular weight moleculesaggregate with NR-LU-13-avidin, the aggregated complexes are readilycleared from the circulation via the RES.

EXAMPLE X

Enhancement of Therapeutic Agent Internalization Through AvidinCrosslinking

The ability of multivalent avidin to crosslink two or more biotinmolecules (or chelate-biotin conjugates) is advantageously used toimprove delivery of therapeutic agents. More specifically, avidincrosslinking induces internalization of crosslinked complexes at thetarget cell surface.

Biotinylated NR-CO-04 (lysine) is prepared according to the methodsdescribed in Example IV.A., above. Doxorubicin-avidin conjugates areprepared by standard conjugation chemistry. The biotinylated NR-CO-04 isadministered to a recipient and allowed to clear from the circulation.One of ordinary skill in the art of radioimmunotherapy is readily ableto determine the optimal time for biotinylated NR-CO-04 tumorlocalization and clearance from the circulation. At such time, thedoxorubicin-avidin conjugate is administered to the recipient. Theavidin portion of the doxorubicin-avidin conjugate crosslinks thebiotinylated NR-CO-04 on the cell surface, inducing internalization ofthe complex. Thus, doxorubicin is more efficiently delivered to thetarget cell.

In a first alternative protocol, a standard three-step pretargetingmethodology is used to enhance intracellular delivery of a drug to atumor target cell. By analogy to the description above, biotinylatedNR-LU-05 is administered, followed by avidin (for blood clearance and toform the middle layer of the sandwich at the target cell-boundbiotinylated antibody). Shortly thereafter, and prior to internalizationof the biotinylated NR-LU-05-avidin complex, a methotrexate-biotinconjugate is administered.

In a second alternative protocol, biotinylated NR-LU-05 is furthercovalently linked to methotrexate. Subsequent administration of avidininduces internalization of the complex and enhances intracellulardelivery of drug to the tumor target cell.

In a third alternative protocol, NR-CO-04-avidin is administered to arecipient and allowed to clear from the circulation and localize at thetarget site. Thereafter, a polybiotinylated species (such asbiotinylated poly-L-lysine, as in Example IV.B., above) is administered.In this protocol, the drug to be delivered may be covalently attached toeither the antibody-avidin component or to the polybiotinylated species.The polybiotinylated species induces internalization of the(drug)-antibody-avidin-polybiotin-(drug) complex.

EXAMPLE XI

Synthesis of DOTA-Biotin Conjugates

A. Synthesis of Nitro-Benzyl-DOTA

The synthesis of aminobenzyl-DOTA was conducted substantially inaccordance with the procedure of McMurry et al., Bioconjugate Chem., 3:108-117, 1992. The critical step in the prior art synthesis is theintermolecular cyclization between disuccinimidylN-(tert-butoxycarbonyl)iminodiacetate and N-(2-aminoethyl)-4-nitrophenylalaninamide to prepare1-(tert-butoxycarbonyl)-5-(4-nitrobenzyl)-3,6,11-trioxo-1,4,7,10-tetraazacyclododecane.In other words, the critical step is the intermolecular cyclizationbetween the bis-NHS ester and the diamine to give the cyclized dodecane.McMurry et al. conducted the cyclization step on a 140 mmol scale,dissolving each of the reagents in 100 ml DMF and adding via a syringepump over 48 hours to a reaction pot containing 4 liters dioxane.

A 5× scale-up of the McMurry et al. procedure was not practical in termsof reaction volume, addition rate and reaction time. Process chemistrystudies revealed that the reaction addition rate could be substantiallyincreased and that the solvent volume could be greatly reduced, whilestill obtaining a similar yield of the desired cyclization product.Consequently on a 30 mmol scale, each of the reagents was dissolved in500 ml DMF and added via addition funnel over 27 hours to a reaction potcontaining 3 liters dioxane. The addition rate of the method employedinvolved a 5.18 mmol/hour addition rate and a 0.047M reactionconcentration.

B. Synthesis of a D-Alanine-Linked Conjugate with a Preserved BiotinCarboxy Moiety

A reaction scheme to form a compound of the following formula isdiscussed below. ##STR42##

The D-alanine-linked conjugate was prepared by first coupling D-alanine(Sigma Chemical Co.) to biotin-NHS ester. The resultantbiotinyl-D-alanine was then activated with1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCI) andN-hydroxysuccinimide (NHS). This NHS ester was reacted in situ withDOTA-aniline to give the desired product which was purified bypreparative HPLC.

More specifically, a mixture of D-alanine (78 mg, 0.88 mmol, 1.2equivalents), biotin-NHS ester (250 mg, 0.73 mmol, 1.0 equivalent),triethylamine (0.30 ml, 2.19 mmol, 3.0 equivalents) in DMF (4 ml) washeated at 110° C. for 30 minutes. The solution was cooled to 23° C. andevaporated. The product solid was acidified with glacial acetic acid andevaporated again. The product biotinyl-D-alanine, a white solid, wassuspended in 40 ml of water to remove excess unreacted D-alanine, andcollected by filtration. Biotinyl-D-alanine was obtained as a whitesolid (130 mg, 0.41 mmol) in 47% yield.

NHS (10 mg, 0.08 mmol) and EDCI (15 mg, 0.07 mmol) were added to asolution of biotinyl-D-alanine (27 mg, 0.08 mmol) in DMF (1 ml). Thesolution was stirred at 23° C. for 60 hours, at which time TLC analysisindicated conversion of the carboxyl group to the N-hydroxy succinimidylester. Pyridine (0.8 ml) was added followed by DOTA-aniline (20 mg, 0.04mmol). The mixture was heated momentarily at approximately 100° C., thencooled to 23° C. and evaporated. The product,DOTA-aniline-D-alanyl-biotinamide was purified by preparative HPLC.

C. Synthesis of N-hydroxyethyl-Linked Conjugate

Iminodiacetic acid dimethyl ester is condensed with biotin-NHS-ester togive biotinyl dimethyl iminodiacetate. Hydrolysis with one equivalent ofsodium hydroxide provides the monomethyl ester after purification fromunder and over hydrolysis products. Reduction of the carboxyl group withborane provides the hydroxyethyl amide. The hydroxyl group is protectedwith t-butyl-dimethyl-silylchloride. The methyl ester is hydrolysed,activated with EDCI and condensed with DOTA-aniline to form the finalproduct conjugate.

D. Synthesis of N-Me-LC-DOTA-Biotin

A reaction scheme is shown below. ##STR43##

Esterification of 6-Aminocaproic acid (Sigma Chemical Co.) was carriedout with methanolic HCl. Trifluoroacetylation of the amino group usingtrifluoroacetic anhydride gave N-6-(methylcaproyl)trifluoroacetamide.The amide nitrogen was methylated using sodium hydride and iodomethanein tetrahydrofuran. The trifluoroacetyl protecting group was cleaved inacidic methanol to give methyl 6-methylamino-caproate hydrochloride. Theamine was condensed with biotin-NHS ester to give methylN-methyl-caproylamido-biotin. Saponification afforded the correspondingacid which was activated with EDCI and NHS and, in situ, condensed withDOTA-aniline to give DOTA-benzylamido-N-methyl-caproylamido-biotin.

1. Preparation of methyl 6-aminocaproate hydrochloride

Hydrogen chloride (gas) was added to a solution of 20.0 g (152 mmol) of6-aminocaproic acid in 250 ml of methanol via rapid bubbling for 2-3minutes. The mixture was stirred at 15°-25° C. for 3 hours and thenconcentrated to afford 27.5 g of the product as a white solid (99%):

H-NMR (DMSO) 9.35 (1 H, broad t), 3.57 (3H, s), 3.14 (2H, quartet), 2.28(2H, t), 1.48 (4H, multiplet), and 1.23 ppm (2H, multiplet).

2. Preparation of N-6-(methylcaproyl)trifluoroacetamide

To a solution of 20.0 g (110 mmol) of methyl 6-aminocaproatehydrochloride in 250 ml of dichloromethane was added 31.0 ml (22.2 mmol)of triethylamine. The mixture was cooled in an ice bath andtrifluoroacetic anhydride (18.0 ml, 127 mmol) was added over a period of15-20 minutes. The mixture was stirred at 0°-10° C. for 1 hour andconcentrated. The residue was diluted with 300 ml of ethyl acetate andsaturated aqueous sodium bicarbonate (3×100 ml). The organic phase wasdried over anhydrous magnesium sulfate, filtered and concentrated toafford 26.5 g of the product as a pale yellow oil (100%):

H-NMR (DMSO) 3.57 (3H, s), 3.37 (2H, t), 3.08 (1.9H, quartet, N--CH₃),2.93 (1.1H, s, N--CH₃), 2.30 (2H, t), 1.52 (4H, multiplet), and 1.23 ppm(2H, multiplet).

3. Preparation of methyl 6-N-methylamino-caproate hydrochloride

To a solution of 7.01 g (29.2 mmol) ofN-6-(methylcaproyl)-trifluoroacetamide in 125 ml of anhydroustetrahydrofuran was slowly added 1.75 g of 60% sodium hydride (43.8mmol) in mineral oil. The mixture was stirred at 15°-25° C. for 30minutes and then 6.2 g (43.7 mmol) of iodomethane was added. The mixturewas stirred at 15°-25° C. for 17 hours and then filtered through celite.The solids were rinsed with 50 ml of tetrahydrofuran. The filtrates werecombined and concentrated. The residue was diluted with 150 ml of ethylacetate and washed first with 5% aqueous sodium sulfite (2×100 ml) andthen with 100 ml of 1N aqueous hydrochloric acid. The organic phase wasdried over anhydrous magnesium sulfate, filtered and concentrated toafford a yellow oily residue. The residue was diluted with 250 ml ofmethanol and then hydrogen chloride (gas) was rapidly bubbled into themixture for 2-3 minutes. The resultant mixture was refluxed for 18hours, cooled and concentrated. The residue was diluted with 150 ml ofmethanol and washed with hexane (3 ×150 ml) to remove mineral oilpreviously introduced with NaH. The methanol phase was concentrated toafford 4.91 g of the product as a yellow oil (86%):

H-NMR (DMSO) 8.80 (2H, broad s), 3.58 (3H, s), 2.81 (2H, multiplet),2.48 (3H, s), 2.30 (2H, t), 1.52 (4H, multiplet), and 1.29 ppm (2H,multiplet).

4. Preparation of methyl 6-(N-methylcaproylamido-biotin

N-hydroxysuccinimidyl biotin (398 mg, 1.16 mmol) was added to a solutionof methyl 6-(N-methyl) aminocaproate hydrochloride (250 mg, 1.28 mmol)in DMF (4.0 ml) and triethylamine (0.18 ml, 1.28 mmol). The mixture washeated in an oil bath at 100° C. for 10 minutes. The solution wasevaporated, acidified with glacial acetic acid and evaporated again. Theresidue was chromatographed on a 25 mm flash chromatography columnmanufactured by Ace Glass packed with 50 g silica (EM Science,Gibbstown, N.J., particle size 0.40-0.63 mm) eluting with 15%MeOH/EtOAc. The product was obtained as a yellow oil (390 mg) in 79%yield.

5. Preparation of 6-(N-methyl-N-biotinyl) amino caproic acid

To a solution of methyl 6-(N-methyl-caproylamido-biotin (391 mg, 1.10mmol) in methanol (2.5 ml) was added a 0.95N NaOH solution (1.5 ml).This solution was stirred at 23° C. for 3 hours. The solution wasneutralized by the addition of 1.0M HCl (1.6 ml) and evaporated. Theresidue was dissolved in water, further acidified with 1.0M HCl (0.4 ml)and evaporated. The gummy solid residue was suspended in water andagitated with a spatula until it changed into a white powder. The powderwas collected by filtration with a yield of 340 mg. 6. Preparation ofDOTA-benzylamido-N-methyl-caproylamido-biotin

A suspension of 6-(N-methyl-N-biotinyl)amino caproic acid (29 mg, 0.08mmol) and N-hydroxysuccinimide (10 mg, 0.09 mmol) in DMF (0.8 ml) washeated over a heat gun for the short time necessary for the solids todissolve. To this heated solution was added EDCI (15 mg, 0.08 mmol). Theresultant solution was stirred at 23° C. for 20 hours. To this stirredsolution were added aminobenzyl-DOTA (20 mg, 0.04 mmol) and pyridine(0.8 ml). The mixture was heated over a heat gun for 1 minute. Theproduct was isolated by preparative HPLC, yielding 3 mg.

E. Synthesis of a bis-DOTA Conjugate with a Preserved Biotin CarboxyGroup

A reaction scheme is shown below. ##STR44##

1. Preparation of methyl 6-bromocaproate (methyl 6-bromohexanoate)

Hydrogen chloride (gas) was added to a solution of 5.01 g (25.7 mmol) of6-bromocaproic acid in 250 ml of methanol via vigorous bubbling for 2-3minutes. The mixture was stirred at 15°-25° C. for 3 hours and thenconcentrated to afford 4.84 g of the product as a yellow oil (90%):

H-NMR (DMSO) 3.58 (3H, s), 3.51 (2H, t), 2.29 (2H, t), 1.78 (2H,pentet), and 1.62-1.27 ppm (4H, m).

2. Preparation of N, N-bis- (methyl 6-hexanoyl ) amine hydrochloride. Toa solution of 4.01 g (16.7 mmol) of N-(methyl6-hexanoyl)-trifluoroacetamide (prepared in accordance with section D.2.herein) in 125 ml of anhydrous tetrahydrofuran was added 1.0 g (25 mmol)of 60% sodium hydride in mineral oil. The mixture was stirred at 15°-25°C. for 1 hour and then 3.50 g (16.7 mmol) of methyl 6-bromocaproate wasadded and the mixture heated to reflux. The mixture was stirred atreflux for 22 hours. NMR assay of an aliquot indicated the reaction tobe incomplete. Consequently, an additional 1.00 g (4.8 mmol) of methyl6-bromocaproate was added and the mixture stirred at reflux for 26hours. NMR assay of an aliquot indicated the reaction to be incomplete.An additional 1.0 g of methyl 6-bromocaproate was added and the mixturestirred at reflux for 24 hours. NMR assay of an aliquot indicated thereaction to be near complete. The mixture was cooled and then directlyfiltered through celite. The solids were rinsed with 100 ml oftetrahydrofuran. The filtrates were combined and concentrated. Theresidue was diluted with 100 ml of methanol and washed with hexane(3×100 ml) to remove the mineral oil introduced with the sodium hydride.The methanol phase was treated with 6 ml of 10N aqueous sodium hydroxideand stirred at 15°-25° C. for 3 hours. The mixture was concentrated. Theresidue was diluted with 100 ml of deionized water and acidified to pH 2with concentrated HCl. The mixture was washed with ether (3×100 ml). Theaqueous phase was concentrated, diluted with 200 ml of dry methanol andthen hydrogen chloride gas was bubbled through the mixture for 2-3minutes. The mixture was stirred at 15°-25° C. for 3 hours and thenconcentrated. The residue was diluted with 50 ml of dry methanol andfiltered to remove inorganic salts. The filtrate was concentrated toafford 1.98 g of the product as a white solid (38%):

H-NMR (DMSO) 8.62 (2H, m) 3.58 (6H, s), 2.82 (4H, m) 2.30 (4H, t),1.67-1.45 (8H, m) and 1.38-1.22 ppm (4H, m).

3. Preparation of N,N'-bis-(methyl 6-hexanol)biotinamide

To a solution of 500 mg (1.46 mmol) of N-hydroxysuccinimidyl biotin in15 ml of dry dimethylformamide was added 600 mg (1.94 mmol) ofN,N-bis-(methyl 6-hexanoyl)amine hydrochloride followed by 1.0 ml oftriethylamine. The mixture was stirred at 80°-85° C. for 3 hours andthen cooled and concentrated. The residue was chromatographed on silicagel, eluting with 20% methanol/ethyl acetate, to afford 620 mg of theproduct as a near colorless oil (85%):

H-NMR (CDCl₃) 5.71 (1H, s), 5.22 (1H, s), 4.52 (1H, m), 4.33 (1H, m),3.60 (3H, s), 3.58 (3H, s), 3.34-3.13 (5H, m), 2.92 (1H, dd), 2.75 (1H,d), 2.33 (6H, m) and 1.82-1.22 ppm (18H, m); TLC-R_(f) 0.39 (20: 80methanol/ethyl acetate).

4. Preparation of N,N-bis-(6-hexanoyl)biotinamide

To a solution of 610 mg (0.819 mmol) of N,N-bis-(methyl6-hexanoyl)-biotinamide in 35 ml of methanol was added 5.0 ml of 1Naqueous sodium hydroxide. The mixture was stirred at 15°-25° C. for 4.5hours and then concentrated. The residue was diluted with 50 ml ofdeionized water acidified to pH 2 with 1N aqueous hydrochloric acid at4° C. The product, which precipitated out as a white solid, was isolatedby vacuum filtration and dried under vacuum to afford 482 mg (84%):

H-NMR (DMSO) 6.42 (1H, s), 6.33 (1H, s), 4.9 (1H, m), 4.12 (1H, m),3.29-3.04 (5H, m), 2.82 (1H, dd), 2.57 (1H, d), 2.21 (6H, m) and1.70-1.10 ppm (18H, m).

5. Preparation of N',N'-bis-(N-hydroxysuccinimidyl6-hexanoyl)-biotinamide

To a solution of 220 mg (0.467 mmol) of N,N-bis-(6-hexanoyl)-biotinamidein 3 ml of dry dimethylformamide was added 160 mg (1.39 mmol) ofN-hydroxysuccinimide followed by 210 mg (1.02 mmol) ofdicyclohexyl-carbodiimide. The mixture was stirred at 15°-25° C. for 17hours and then concentrated. The residue was chromatographed on silicagel, eluting with 0.1: 20: 80 acetic acid/methanol/ethyl acetate, toafford 148 mg of the product as a foamy off-white solid (48%):

H-NMR (DMSO) 6.39 (1H, s), 6.32 (1H, s), 4.29 (1H, m), 4.12 (1H, m),3.30-3.03 (5H, m), 2.81 (9H, dd and s), 2.67 (4H, m), 2.57 (1H, d), 2.25(2H, t), 1.75-1.20 (18H, m); TLC-R_(f) 0.37 (0.1:20:80 aceticacid/methanol/ethyl acetate).

6. Preparation of N,N-bis-(6-hexanoylamidobenzyl-DOTA)-biotinamide

To a mixture of 15 mg of DOTA-benzylamine and 6 0 mg ofN',N'-bis-(N-hydroxysuccinimidyl 6-hexanoyl)-biotinamide in 1.0 ml ofdry dimethylformamide was added 0.5 ml of dry pyridine. The mixture wasstirred at 45°-50° C. for 4.5 hours and at 15°-25° C. for 12 hours. Themixture was concentrated and the residue chromatographed on a 2.1×2.5 cmoctadecylsilyl (ODS) reverse-phase preparative HPLC column eluting witha--20 minute gradient profile of 0.1:95:5 to 0.1:40:60 trifluoroaceticacid:water:acetonitrile at 13 ml/minute to afford the desired product.The retention time was 15.97 minutes using the aforementioned gradientat a flow rate of 1.0 ml/minute on a 4.6 mm×25 cm ODS analytical HPLCcolumn.

F. Synthesis of an N-methyl-glycine Linked Conjugate

A reaction scheme for this synthesis is shown below. ##STR45##

The N-methyl glycine-linked DOTA-biotin conjugate was prepared by ananalogous method to that used to prepare D-alanine-linked DOTA-biotinconjugates. N-methyl-glycine (trivial name sarcosine, available fromSigma Chemical Co.) was condensed with biotin-NHS ester in DMF andtriethylamine to obtain N-methyl glycyl-biotin. N-methyl-glycyl biotinwas then activated with EDCI and NHS. The resultant NHS ester was notisolated and was condensed in situ with DOTA-aniline and excesspyridine. The reaction solution was heated at 60° C. for 10 minutes andthen evaporated. The residue was purified by preparative HPLC to give[(N-methyl-N-biotinyl)-N-glycyl]-aminobenzyl-DOTA.

1. Preparation of (N-methyl)glycyl biotin

DMF (8.0 ml) and triethylamine (0.61 ml, 4.35 mmol) were added to solidsN-methyl glycine (182 mg, 2.05 mmol) and N-hydroxy-succinimidyl biotin(500 mg, 1.46 mmol). The mixture was heated for 1 hour in an oil bath at85° C. during which time the solids dissolved producing a clear andcolorless solution. The solvents were then evaporated. The yellow oilresidue was acidified with glacial acetic acid, evaporated andchromatographed on a 27 mm column packed with 50 g silica, eluting with30% MeOH/EtOAc 1% HOAc to give the product as a white solid (383 mg) in66% yield.

H-NMR (DMSO): 1.18-1.25 (m, 6H, (CH₂)₃), 2.15, 2.35 (2 t's, 2H, CH₂ CO),2.75 (m, 2H, SCH₂), 2.80, 3.00 (2 s's, 3H, NCH₃), 3.05-3.15 (m, 1H,SCH), 3.95, 4.05 (2 s's, 2H, CH₂ N), 4.15, 4.32 (2 m's, 2H, 2CHN's),6.35 (s, NH), 6.45 (s, NH).

2. Preparation of [(N-methyl-N-biotinyl)glycyl]aminobenzyl-DOTA

N-hydroxysuccinimide (10 mg, 0.08 mmol) and EDCI (15 mg, 6.08 mmol) wereadded to a solution of (N-methylglycyl biotin (24 mg, 0.08 mmol) in DMF(1.0 ml). The solution was stirred at 23 ° C. for 64 hours. Pyridine(0.8 ml) and aminobenzyl-DOTA (20 mg, 0.04 mmol) were added. The mixturewas heated in an oil bath at 63° C. for 10 minutes, then stirred at 23°C. for 4 hours. The solution was evaporated. The residue was purified bypreparative HPLC to give the product as an off white solid (8 mg, 0.01mmol) in 27% yield.

H-NMR (D₂ O): 1.30-1.80 (m, 6H), 2.40, 2.55 (2 t's, 2H, CH₂ CO),2.70-4.2 (complex multiplet), 4.35 (m, CHN), 4.55 (m, CHN), 7.30 (m, 2H,benzene hydrogens), 7.40 (m, 2H, benzene hydrogens).

G. Synthesis of a Short Chain Amine-Linked Conjugate with a ReducedBiotin Carboxy Group

A two-part reaction scheme is shown below. ##STR46##

The biotin carboxyl group is reduced with diborane in THF to give aprimary alcohol. Tosylation of the alcohol with tosyl chloride inpyridine affords the primary tosylate. Aminobenzyl DOTA is acylated withtrifluoroacetic anhydride in pyridine to give(N-trifluoroacetyl)aminobenzyl-DOTA. Deprotonation with 5.0 equivalentsof sodium hydride followed by displacement of the biotin tosylateprovides the(N-trifluoracetamido-N-descarboxylbiotinyl)aminobenzyl-DOTA. Acidiccleavage of the N-trifluoroacetamide group with HCl(g) in methanolprovides the amine-linked DOTA-biotin conjugate.

EXAMPLE XII

Human Clinical Trial: Three-Step Pretargeting

Patients were selected on the basis of a variety of criteria. Thethree-step pretargeting protocol to which such patients were subjectedproceeded as follows:

Step 1--Patients received 10 mg whole NR-LU-10-LC-biotin conjugateprepared in accordance with the procedure described in Example IV. Insome cases, the conjugate was radiolabeled with Tc-99m in accordancewith the procedure referenced above for radiolabeling NR-LU-10 Fab tofacilitate monitoring of the conjugate in vivo. The NR-LU-10-LC-biotinconjugate, either radiolabeled or non-radiolabeled, was diluted in 30 MLof normal saline and administered by intravenous injection over 3-5minutes. The conjugate was administered within 4 hours after completionof the radiolabeling procedure and exhibited 20-25 mCi activity whenadministered.

Step 2--Avidin was administered intravenously 24-36 hours afteradministration of the NR-LU-10-LC-biotin conjugate. The avidinadministration was conducted in two stages: 3-5 mg in 5 mL ofphysiological solution as a rapid bolus dose and 40-80 mg in 100 mL ofphysiological solution 30 minutes later. Some patients receivedradiolabeled avidin (10-15 mCi activity at the time of administration)to facilitate in vivo monitoring of this component of the three-steppretargeting system. Avidin was also radiolabeled substantially inaccordance with the procedure referenced above for NR-LU-10 Fabradiolabeling.

Step 3--Diethylenetriaminepentacetic acid-alpha, W-bis(biocytinamide)(DTPA-bis-biotin, available from Sigma Chemical Company, St. Louis, Mo.)was radiolabeled with In-111 as set forth below. DTPA-biotin was dilutedin PBS, pH 7.4, to a concentration of 2 micrograms/microliter. Thesolution was sterilized by 0.22 mm millipore filtration. ¹¹¹ InCl₃ wasdiluted in citrate buffer (0.02M; pH 6.5) to 740 kBq/μl. The tworeagents were mixed and allowed to react at room temperature for 10minutes. Generally, a 98% chelation of In-111 to DTPA-biotin wasachieved, as verified by paper chromatography. Administration of 2-5 mgof In-111-DTPA-biotin (5-10 mCi activity) diluted in 5 mL of saline wasconducted intravenously, over 1 minute, 24 hours after avidinadministration. The patients were evaluated for 24 hours followingadministration of In-111-DTPA-biotin.

A 66 year old male presented with a large primary lesion in theascending colon and a small lesion in the transverse colon (polyp). Thispatient was subjected to a three-step pretargeting protocol as follows:

t=0; 10 mg monoclonal antibody-biotin

t=25 hour; 10 mg avidin;

t=25.3 hour; 90 mg avidin; and

t=24 hour; 6 mCi In-111-DTPA-biotin.

Images were taken and analyzed by the attending physician. The largelesion was visualized in a 2 hour SPECT image.

EXAMPLE XIII

Three-Step Pretargeting Using Y-90

A patient presents with ovarian cancer. A monoclonal antibody (MAb)directed to an ovarian cancer cell antigen, e.g., NR-LU-10, isconjugated to biotin to form a MAb-biotin conjugate. The MAb-biotinconjugate is administered to the patient in an amount sufficient tosubstantially saturate the available antigenic sites at the target(which amount is at least sufficient to allow the capture of atherapeutically effective radiation dose at the target and which amountmay be in excess of the maximum tolerated dose of conjugateadministrable in a targeted, chelate-labeled molecule protocol, such asadministration of monoclonal antibody-chelate-radionuclide conjugate).The MAb-biotin so administered is permitted to localize to target cancercells for 24-48 hours. Next, an amount of avidin sufficient to clearnon-targeted MAb-biotin conjugate and to bind to the targeted biotin isadministered.

A biotin-radionuclide chelate conjugate of the type discussed in ExampleXI(F) above is radiolabeled with Y-90 as set forth below. Carrier free⁹⁰ YCl₃ (available from NEN-DuPont, Wilmington, Del.) at 20-200 μl in0.05N HCl was diluted with ammonium acetate buffer (0.SM, pH 5) to atotal volume of 0.4 ml. 50 μl (500 mg/ml) of ascorbic acid and 50-100 μl(10 mg/ml) of DOTA-biotin were added to the buffered ⁹⁰ YCl₃ solution.The mixture was incubated for one hour at 80° C. Upon completion of theincubations, 55 μl of 100 mM DTPA was added to the mixture to chelateany unbound ⁹⁰ Y. The final preparation was diluted to 10 ml with 0.9%NaCl.

The radiolabeled DOTA-biotin conjugate is administered to the patient ina therapeutically effective dose. The biotin-radionuclide chelateconjugate localizes to the targeted MAb-biotin-avidin moiety or issubstantially removed from the patient via the renal pathway.

Kits containing one or more of the components described above are alsocontemplated. For instance, radiohalogenated biotin may be provided in asterile container for use in pretargeting procedures. A chelate-biotinconjugate provided in a sterile container is suitable forradiometallation by the consumer; such kits would be particularlyamenable for use in pretargeting protocols. Alternatively,radiohalogenated biotin and a chelate-biotin conjugate may be vialed ina non-sterile condition for use as a research reagent.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. An improved method of enhancing active agentlocalization at a target site in a mammalian recipient, which methodcomprises:administering to the recipient a first conjugate comprising anantibody or antigen-binding antibody fragment and a biotin, whereuponthe first conjugate localizes to the target site; administering to therecipient avidin or streptavidin; and thereupon administering to therecipient a second conjugate comprising biotin, a linker resistant tobiotinidase cleavage and an active agent, wherein second agentlocalization at the target site is enhanced as a result of priorlocalization of the first conjugate and wherein the improvement is thatthe second conjugate comprises a biotinidase-resistant biotin-DOTAcompound of the following formula: ##STR47## wherein a linker L isselected from the group consisting of: 1) a D-amino acid-containinglinker of the formula ##STR48## 2) a linker of the formula ##STR49## 3)a linker of the formula ##STR50## 4) a linker of the formula ##STR51##wherein L' is selected from the group consisting of: a)--NH--CO--(CH₂)_(n) --O; b) --NH--; ##STR52## d) --NH--CS--NH--; and e)--NH--CO--(CH₂)_(n) --NH--,wherein R¹ is hydrogen, lower alkyl; loweralkyl substituted with one or more hydrophilic groups selected from thegroup consisting of (CH₂)_(m) --OH, (CH₂)_(m) --OSO₃, (CH₂)_(m) --SO₃,##STR53## where m is 1 or 2; glucuronide-substituted amino acids; andother glucuronide derivatives; R² is hydrogen; lower alkyl; substitutedlower alkyl having one or more substituents selected from the groupconsisting of hydroxy, sulfate, and phosphonate; or a hydrophilicmoiety; R³ is hydrogen; an amine; a lower alkyl; a hydroxy-, sulfate- orphosphonate-substituted lower alkyl; a glucuronide; or aglucuronide-derivatized amino acid; R⁴ is hydrogen, lower alkyl or##STR54## R' is hydrogen; --(CH₂)₂ --OH or a sulfate or phosphonatederivative thereof; or ##STR55## R" is a bond or --(CH₂)_(n) --CO--NH--;and n ranges from 0-5, wherein R³ and R⁴ cannot both be hydrogen andwherein the active agent is a radionuclide.
 2. A method of claim 1wherein the targeting moiety is a monoclonal antibody, or a monovalentfragment thereof.
 3. A method of claim 2 wherein the monoclonal antibodyis a human, a humanized or a chimeric monoclonal antibody.
 4. A methodof claim 2 wherein the monoclonal antibody or fragment thereof isreactive with an antigen recognized by the antibody NR-LU-10.
 5. Amethod of claim 1 wherein the active agent is a radionuclide selectedfrom the group consisting of Re-186, Re-188, Tc-99m, Y-90, At-211,Pb-212, Bi-212, Sm-153, Eu-169, Lu-177, Cu-67, Rh-105, In-111, Au-198,I-123 and I-131.
 6. A method of claim 1 wherein the step ofadministering the second conjugate is conducted by intralesional orintraarterial injection.
 7. A method of claim 6 wherein the secondconjugate is administered via an artery supplying target site tissue. 8.A method of claim 6 wherein the second conjugate is administered via anartery selected from the group consisting of hepatic artery, carotidartery, bronchial artery and renal artery.
 9. A method of claim 1wherein the second conjugate is administered intravenously.
 10. A methodof claim 1 wherein L is a D-amino acid-incorporating linker of theformula ##STR56##
 11. A method of claim 10 wherein R¹ is CH₃ and R² isH.
 12. A method of claim 1 wherein L is a linker of the formula##STR57##
 13. A method of claim 12 wherein R³ is hydrogen; R⁴ is CH₃ ;and n is
 4. 14. A method of claim 12 wherein R³ is hydrogen; R⁴ is CH₃ ;and n is
 0. 15. A method of claim 12 wherein R³ is hydrogen; R⁴ is##STR58## and n is
 4. 16. A method of claim 1 wherein L is a linker ofthe formula ##STR59## wherein L' is selected from the group consistingof: a) --NH--CO--(CH₂)_(n) --O--;b) --NH--; ##STR60## d) --NH--CS--NH--;and e) --NH--CO--(CH₂)_(n) --NH-- or a bis-DOTA derivative thereof. 17.An improved method of enhancing active agent localization at a targetsite in a mammalian recipient, which method comprises:administering tothe recipient a first conjugate comprising an antibody or anantigen-binding antibody fragment thereof and a biotin, whereupon thefirst conjugate localizes to the target site; administering to therecipient avidin or streptavidin; and thereupon administering to therecipient a second conjugate comprising biotin, a linker resistant tobiotinidase cleavage and an active agent, wherein second agentlocalization at the target site is enhanced as a result of priorlocalization of the first conjugate and wherein the improvement is thatthe second conjugate comprises a biotinidase-resistant biotin-DOTAcompound of the following formula: ##STR61## and further wherein thelinker L has the formula: ##STR62## wherein R³ is hydrogen, R⁴ is CH₃and n is 4; R³ is hydrogen, R⁴ is CH₃ and n is 0; or R³ is hydrogen, R⁴is ##STR63## and n is 4; or R³ is hydrogen; R⁴ is ##STR64## and n is 0,and wherein the active agent is a radionuclide.
 18. An improved methodof enhancing active agent localization at a target site in a mammalianrecipient, which method comprises;administering to the recipient a firstconjugate comprising an antibody or antigen-binding antibody fragmentthereof and a biotin, whereupon the first conjugate localizes to thetarget site; administering to the recipient avidin or streptavidin; andthereupon administering to the recipient a second conjugate comprisingbiotin, a linker resistant to biotinidase cleavage and an active agent,wherein second agent localization at the target site is enhanced as aresult of prior localization of the first conjugate and wherein theimprovement is that the second conjugate comprises abiotinidase-resistant biotin-DOTA compound of the following formula:##STR65## and further wherein the linker L is a D-amino acidincorporating a linker of the formula ##STR66## wherein R¹ is CH₃ and R²is H, and wherein the active agent is a radionuclide.